Biochem-handout

Ross University School of Medicine

Course Handout

Biochemistry and Genetics Department of Biochemistry Drs Blanchetot, Beevers, Buxbaum, Grogan, James, Larsen, LaVille, Meisenberg, Sands, Smolanoff and Mrs Lambert

© DeVry Inc. 2007–2009

This text is © DeVry Inc. 2007–2009. It may be freely used for academic purposes as long as it is kept unchanged and complete, including this copyright notice. All trademarks are acknowledged as trademarks of their respective owners. Please report any errors found in this text or any suggestions for its improvement to the maintainer ([email protected]).

This text was created with LATEX using the MikTEX system (http://www.dante.de). Chemical structures were created with ISIS-Draw (http://www.mdli.com). Gnuplot (http://www.cs.dartmouth.edu/gnuplot_info.html) was used for plotting mathematical functions. For conversion between graphic formats the Gnu Image Manipulation Package (Gimp, http://www.xcf.berkely.edu/∼gimp/gimp.html) was used. Molecular structures were created with Deepview (http://www.expasy.ch/spdbv/mainpage.html) from PDBfiles (http://bip.weizmann.ac.il/oca-bin/ocamain). Three-dimensional graphics were rendered with the Persistence Of Vision Ray-tracer (POV-Ray, http://www.povray.org). Many thanks to all those who made these free tools available on the net.

Contents Welcome

I.

xix

Semester one, Mini I

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1. Introduction to Biomolecules 1.1. Elements and molecules . . . . . . . . . . . . . 1.2. Covalent Bonds And Non-covalent Interactions 1.2.1. Covalent bonds . . . . . . . . . . . . . . 1.2.2. Non-covalent interactions . . . . . . . . 1.3. Bonds in biomolecules . . . . . . . . . . . . . . 1.4. Isomers . . . . . . . . . . . . . . . . . . . . . . 1.5. Acids and bases . . . . . . . . . . . . . . . . . . 1.6. Fats and carbohydrates . . . . . . . . . . . . . 1.7. Objectives in summary . . . . . . . . . . . . . . 1.7.1. Molecular Structure . . . . . . . . . . .

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3 3 3 3 4 5 8 9 11 13 13

2. Energy changes and rates of chemical reactions 2.1. Thermodynamics . . . . . . . . . . . . . . . . 2.2. Reaction kinetics . . . . . . . . . . . . . . . . 2.2.1. Order of Reactions . . . . . . . . . . . 2.2.2. The principle of Le Chatelier . . . 2.3. Catalysis . . . . . . . . . . . . . . . . . . . . . 2.4. Example questions . . . . . . . . . . . . . . . 2.5. Objectives . . . . . . . . . . . . . . . . . . . .

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3. Amino acids and proteins 3.1. Amino acids . . . . . . . . . . . . . . . . 3.1.1. General structure of amino acids 3.1.2. The 22 amino acids in proteins . 3.1.3. The pI-value . . . . . . . . . . . 3.1.4. The one-letter code . . . . . . . . 3.2. Proteins . . . . . . . . . . . . . . . . . . 3.2.1. The peptide bond . . . . . . . . .

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3.2.2. Protein structure . . . . . . . 3.2.3. Proteins in the laboratory . . 3.3. Protein folding diseases . . . . . . . 3.3.1. Spongiform encephalopathies 3.3.2. Morbus Alzheimer . . . . . 3.3.3. Morbus Parkinson . . . . . 3.3.4. Chorea Huntington . . . . 3.4. Example questions . . . . . . . . . . 3.5. Objectives . . . . . . . . . . . . . . .

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4. DNA and Gene Expression 4.1. DNA Structure . . . . . . . . . . . . . . . . 4.1.1. Bases, Nucleosides and Nucleotides . 4.1.2. The DNA Double-Helix. . . . . . . . 4.1.3. Chemical stability . . . . . . . . . . 4.1.4. Supercoiling . . . . . . . . . . . . . . 4.2. DNA Replication . . . . . . . . . . . . . . . 4.2.1. Semi-conservative replication . . . . 4.2.2. DNA polymerases . . . . . . . . . . 4.2.3. Steps in bacterial DNA replication . 4.3. RNA and Transcription . . . . . . . . . . . 4.3.1. RNA Structure . . . . . . . . . . . . 4.3.2. Types of RNA . . . . . . . . . . . . 4.3.3. Transcription . . . . . . . . . . . . . 4.3.4. Post-transcriptional processing . . . 4.4. Protein Synthesis . . . . . . . . . . . . . . . 4.4.1. The Genetic Code . . . . . . . . . . 4.4.2. tRNA . . . . . . . . . . . . . . . . . 4.4.3. Ribosomes . . . . . . . . . . . . . . . 4.4.4. Steps in translation . . . . . . . . . . 4.4.5. Antibiotics . . . . . . . . . . . . . . 4.5. Regulation of Gene Expression . . . . . . . 4.6. Virus . . . . . . . . . . . . . . . . . . . . . . 4.6.1. Virus Structure . . . . . . . . . . . . 4.6.2. The Lytic Cycle of Bacteriophage T4 4.6.3. The Lysogenic Cycle of λ phage . . . 4.6.4. Animal Virus . . . . . . . . . . . . . 4.6.5. RNA Virus . . . . . . . . . . . . . . 4.6.6. Retrovirus . . . . . . . . . . . . . . . 4.6.7. Plasmids . . . . . . . . . . . . . . . . 4.7. Genetic Recombination . . . . . . . . . . . .

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Contents

4.8. Types of Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 4.8.1. Parasexual Processes in Bacteria . . . . . . . . . . . . . . . . . . . . 105 4.9. Objectives in Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 5. The Human Genome and Mutations 5.1. The Human Genome . . . . . . . . . . . . 5.2. Chromatin Structure . . . . . . . . . . . . 5.2.1. Histones and Nucleosomes . . . . . 5.2.2. Repetitive DNA . . . . . . . . . . 5.2.3. Mobile DNA . . . . . . . . . . . . 5.2.4. Genes . . . . . . . . . . . . . . . . 5.2.5. Telomeres . . . . . . . . . . . . . . 5.2.6. DNA Replication . . . . . . . . . . 5.3. Mutations . . . . . . . . . . . . . . . . . . 5.3.1. Types of Mutation . . . . . . . . . 5.3.2. Causes of Mutations . . . . . . . . 5.3.3. Mutagenesis Testing . . . . . . . . 5.4. DNA Repair . . . . . . . . . . . . . . . . . 5.4.1. Repair Defects . . . . . . . . . . . 5.5. Eukaryotic Gene Expression . . . . . . . . 5.5.1. Transcription . . . . . . . . . . . . 5.5.2. mRNA Processing . . . . . . . . . 5.5.3. Translation . . . . . . . . . . . . . 5.6. Regulation of Eukaryotic Gene Expression 5.7. Objectives in Summary . . . . . . . . . .

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109 109 110 110 110 111 111 112 112 112 113 113 114 114 115 116 116 116 117 117 119

6. Chromosome Aberrations 6.1. The Human Karyotype . . . . . . . . . . . . . . . . 6.2. Sex Chromatin . . . . . . . . . . . . . . . . . . . . 6.3. Types Of Chromosome Aberrations . . . . . . . . . 6.4. Autosomal Trisomies . . . . . . . . . . . . . . . . . 6.4.1. Trisomy 21 (Down syndrome) . . . . . . . 6.4.2. Trisomy 18 (Edward syndrome) . . . . . . 6.4.3. Trisomy 13 (Patau syndrome) . . . . . . . 6.5. Sex Chromosome Aberrations: Male . . . . . . . . 6.5.1. Klinefelter syndrome . . . . . . . . . . . 6.5.2. XYY constitution (“murderer chromosome”) 6.5.3. XX Males . . . . . . . . . . . . . . . . . . . 6.6. Sex Chromosome Aberrations: Female . . . . . . . 6.6.1. Turner’s syndrome (gonadal dysgenesis) . 6.6.2. Triple-X (47, XXX, “superfemale”) . . . . . 6.6.3. XY females . . . . . . . . . . . . . . . . . .

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Contents

6.7. Abnormal Sexual Development With Normal Chromosomes 6.7.1. True hermaphroditism . . . . . . . . . . . . . . . . . 6.7.2. Mixed Gonadal Dysgenesis . . . . . . . . . . . . . . 6.7.3. Female Pseudohermaphroditism. . . . . . . . . . . . 6.7.4. Male Pseudohermaphroditism . . . . . . . . . . . . . 6.7.5. Chromosomal Rearrangements . . . . . . . . . . . . 6.8. Objectives in Summary . . . . . . . . . . . . . . . . . . . . 7. Enzymes 7.1. History of enzymology . . . . . . . . . . . . . . . . . 7.2. Classification of enzymes . . . . . . . . . . . . . . . . 7.2.1. Systematic name . . . . . . . . . . . . . . . . 7.2.2. Enzyme classes and EC codes . . . . . . . . . 7.3. Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.1. The Henri-Michaelis-Menten (HMM)-equation 7.3.2. Catalytic perfection . . . . . . . . . . . . . . 7.3.3. Environmental influences on enzyme activity 7.3.4. Cooperativity . . . . . . . . . . . . . . . . . . 7.4. Enzyme inhibition . . . . . . . . . . . . . . . . . . . 7.4.1. Competitive inhibition . . . . . . . . . . . . . 7.4.2. Uncompetitive inhibition . . . . . . . . . . . . 7.4.3. Noncompetitive inhibition . . . . . . . . . . . 7.4.4. Mixed inhibition . . . . . . . . . . . . . . . . 7.5. Enzyme inactivation . . . . . . . . . . . . . . . . . . 7.6. Enzymes with multiple substrates or products . . . . 7.7. How do enzymes do it? . . . . . . . . . . . . . . . . . 7.7.1. Protease reaction mechanism . . . . . . . . . 7.8. Coenzymes . . . . . . . . . . . . . . . . . . . . . . . 7.8.1. Adenosine Triphosphate (ATP) . . . . . . . . 7.8.2. Redox Coenzymes . . . . . . . . . . . . . . . 7.8.3. Other Coenzymes . . . . . . . . . . . . . . . . 7.9. Enzymes in clinical diagnostics . . . . . . . . . . . . 7.10. Membrane Transport . . . . . . . . . . . . . . . . . . 7.10.1. Passive transport . . . . . . . . . . . . . . . . 7.10.2. Active transport . . . . . . . . . . . . . . . . 7.11. Homeostasis of the Intracellular Environment . . . . 7.11.1. Ion concentrations . . . . . . . . . . . . . . . 7.12. Useful web resources . . . . . . . . . . . . . . . . . . 7.13. Example questions . . . . . . . . . . . . . . . . . . . 7.14. Objectives . . . . . . . . . . . . . . . . . . . . . . . .

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Contents

8. Methods in Molecular Medicine 8.1. Restriction Endonucleases . . . . . . . . . . . . . . . . 8.2. Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Southern Blotting . . . . . . . . . . . . . . . . . . . 8.4. The Polymerase Chain Reaction (PCR) . . . . . . . . 8.5. DNA Sequencing and Deduced Functions . . . . . . . 8.6. Gene Mapping . . . . . . . . . . . . . . . . . . . . . . 8.6.1. Fluorescent in-situ Hybridization (FISH) . . . . 8.6.2. Deletion Mapping . . . . . . . . . . . . . . . . 8.6.3. Linkage Analysis . . . . . . . . . . . . . . . . . 8.6.4. Candidate Genes . . . . . . . . . . . . . . . . . 8.7. Cloning and Genomic Libraries . . . . . . . . . . . . . 8.8. cDNA Cloning and Expression Cloning . . . . . . . . . 8.9. Site-Directed Mutagenesis and Protein Engineering . . 8.10. Use of DNA Diagnostics . . . . . . . . . . . . . . . . . 8.10.1. Southern Blotting with Allele-Specific Probes 8.11. Use of PCR. . . . . . . . . . . . . . . . . . . . . . . . . 8.12. Dot-Blotting . . . . . . . . . . . . . . . . . . . . . . . 8.13. OBJECTIVES IN SUMMARY . . . . . . . . . . . . .

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II. Semester one, Mini II 9. Glycolysis: Splitting glucose in half 9.1. Overview . . . . . . . . . . . . 9.2. Glycolysis . . . . . . . . . . . . 9.2.1. Reactions of glycolysis . 9.2.2. Regulation of glycolysis 9.2.3. Inhibition of glycolysis 9.2.4. Anaerobic glycolysis . . 9.3. Objectives in Summary . . . .

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10.Plasma Proteins 10.1. Plasma Proteins: Overview . . . . . . . . . 10.1.1. Functions of plasma proteins . . . . 10.2. Separation of Plasma Proteins . . . . . . . . 10.2.1. Albumin . . . . . . . . . . . . . . . . 10.2.2. Transport Proteins . . . . . . . . . . 10.2.3. Protease Inhibitors . . . . . . . . . . 10.3. Plasma Proteins in Disease States . . . . . . 10.3.1. Plasma Components for Clinical Use 10.4. Clinical Enzymology . . . . . . . . . . . . .

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10.5. Objectives in summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 11.Blood Coagulation 11.1. The Biochemistry of Blood Coagulation . . . . . . . 11.1.1. Primary hemostasis (platelet-plug formation) 11.1.2. Secondary hemostasis (fibrin clot formation) . 11.1.3. Clinical Correlates . . . . . . . . . . . . . . . 11.1.4. Laboratory Tests . . . . . . . . . . . . . . . . 11.1.5. Features of Coagulation . . . . . . . . . . . . 11.1.6. Genetics of Blood Coagulation . . . . . . . . 11.2. Objectives in Brief . . . . . . . . . . . . . . . . . . .

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12.Blood 12.1. Blood Groups . . . . . . . . . . . . . . . . . . . . 12.1.1. The ABO system . . . . . . . . . . . . . . 12.1.2. The Rhesus System . . . . . . . . . . . . . 12.1.3. Other blood group systems . . . . . . . . 12.2. Structure of Hemoglobin . . . . . . . . . . . . . . 12.2.1. Chemical Inactivation of Hemoglobin . . . 12.2.2. Allosteric Properties . . . . . . . . . . . . 12.2.3. The Hemoglobinopathies . . . . . . . . . . 12.3. Objectives in Summary . . . . . . . . . . . . . . 12.3.1. Blood Groups . . . . . . . . . . . . . . . . 12.3.2. Hemoglobin and Myoglobin Biochemistry 12.3.3. Hemoglobinopathies . . . . . . . . . . . .

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219 219 219 220 221 221 221 222 225 228 228 228 229

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III. Semester one, Mini III

231

13.Mendelian Inheritance 13.1. The Patterns of Mendelian Inheritance . . . . 13.2. Variations of gene transmission and expression 13.3. Functional classification of mutations . . . . . . 13.3.1. Loss of function . . . . . . . . . . . . . . 13.3.2. Gain of function . . . . . . . . . . . . . 13.4. Linked Markers for Genotype Prediction . . . . 13.5. Pedigree Analysis . . . . . . . . . . . . . . . . . 13.5.1. X-linked recessive inheritance . . . . . . 13.6. Modified Risk: Bayes Theorem . . . . . . . . . 13.7. Objectives in Summary . . . . . . . . . . . . .

233 233 234 235 236 236 237 238 239 240 241

viii

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14.Krebs- (TCA-) cycle: Burning the carbon skeleton 14.1. The Pyruvate Dehydrogenase Reaction . . . . . . . . . . . . . 14.1.1. The pyruvate dehydrogenase complex . . . . . . . . . 14.1.2. Overall reaction . . . . . . . . . . . . . . . . . . . . . . 14.1.3. Functional impairment . . . . . . . . . . . . . . . . . . 14.2. The TCA Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.1. Reactions . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.2. Products of the TCA cycle . . . . . . . . . . . . . . . 14.2.3. Regulation of pyruvate dehydrogenase and TCA cycle 14.3. Inhibition of the TCA cycle . . . . . . . . . . . . . . . . . . . 14.3.1. Other reactions of TCA cycle intermediates . . . . . . 14.4. Shuttles Across the Inner Mitochondrial Membrane . . . . . . 14.5. Shuttles for electrons from cytoplasmic NADH + H+ . . . . . 14.5.1. Other substrates and products . . . . . . . . . . . . . 14.6. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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243 243 243 243 243 244 244 244 244 245 245 246 246 247 247

15.Respiratory Chain, Oxidative Phosphorylation and Reactive 15.1. Respiratory Chain: Burning Hydrogen . . . . . . . . . . 15.1.1. Overall reactions . . . . . . . . . . . . . . . . . . 15.1.2. The redox potential . . . . . . . . . . . . . . . . 15.1.3. Components of the electron transport chain . . . 15.2. Making ATP from electricity . . . . . . . . . . . . . . . 15.2.1. Phosphorylation sites . . . . . . . . . . . . . . . . 15.2.2. Mechanism of oxidative phosphorylation . . . . . 15.2.3. Energy yield from glucose . . . . . . . . . . . . . 15.2.4. Regulation of electron flow and phosphorylation . 15.2.5. Inhibition of oxidative phosphorylation . . . . . . 15.3. Reactive Oxygen Derivatives . . . . . . . . . . . . . . . . 15.3.1. Protective mechanisms . . . . . . . . . . . . . . . 15.4. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . .

Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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249 249 249 249 250 251 251 252 252 252 252 253 254 255

16.Single-Gene Disorders and Traits 16.1. Skeletal and Connective Tissue Diseases . . 16.2. Skeletal Dysplasias and Dysostoses . . . . . 16.3. Diseases Of Muscles and Peripheral Nerves . 16.3.1. Other Muscular Dystrophies . . . . . 16.3.2. Myotonic Dystrophy . . . . . . . . . 16.3.3. Peripheral Neuropathies . . . . . . . 16.4. CNS Disorders . . . . . . . . . . . . . . . . 16.4.1. Hereditary ataxias . . . . . . . . . . 16.4.2. Mental Retardation . . . . . . . . .

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257 257 258 259 259 260 260 261 261 261

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16.5. Blood Diseases . . . . . . . . . . . . . 16.5.1. Clotting Disorders . . . . . . . 16.5.2. Structural defects of RBCs . . 16.6. Skin Diseases . . . . . . . . . . . . . . 16.6.1. Occulocutaneous albinism . . . 16.6.2. Epidermolysis bullosa (EB) . . 16.7. Polycystic Kidney Disease . . . . . . . 16.8. Cystic Fibrosis . . . . . . . . . . . . . 16.9. Blindness and Deafness . . . . . . . . . 16.10.“Harmless” Mendelian Traits . . . . . . 16.11.Tuberous Sclerosis . . . . . . . . . . . 16.12.Phenylketonuria (PKU) . . . . . . . . 16.13.Hemochromatosis . . . . . . . . . . . . 16.14.Diseases Caused by Unusual Mutations 16.14.1.Imprinting-related Syndromes . 16.14.2.Lepore Hemoglobins . . . . . . 16.15.Objectives in Summary . . . . . . . . 17.Hormone Biochemistry 17.1. Types of Extracellular Messenger 17.2. Hormone Receptors . . . . . . . . 17.3. Types of Hormone Receptor . . . 17.4. Receptors Coupled to G-Protein . 17.5. Cyclic AMP (cAMP) . . . . . . . 17.6. Calcium and Phosphatidylinositol 17.7. Cyclic GMP (cGMP). . . . . . . 17.8. Desensitization of Receptors . . . 17.9. Objectives in Summary . . . . .

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262 262 263 263 263 264 264 264 265 265 266 266 267 268 268 269 269

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271 271 271 272 273 274 275 277 277 278

IV. Semester two, Mini I

279

18.Vitamins and minerals 18.1. Nutrient doses . . . . . . . . . . . . . . . . . . . 18.1.1. The US Recommended Daily Allowance 18.1.2. Assessing nutrient stores and needs . . . 18.2. Vitamins . . . . . . . . . . . . . . . . . . . . . . 18.2.1. Fat soluble vitamins . . . . . . . . . . . 18.2.2. Water soluble vitamins . . . . . . . . . . 18.3. Minerals . . . . . . . . . . . . . . . . . . . . . . 18.3.1. Mass elements . . . . . . . . . . . . . . 18.3.2. Trace elements . . . . . . . . . . . . . .

281 281 283 283 285 286 299 318 319 325

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18.3.3. Ultratrace elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 18.4. Objectives in summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 19.Carbohydrate Metabolism 19.1. Gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.1.1. The first bypass: From pyruvate to phosphoenolpyruvate 19.1.2. The second and third bypasses . . . . . . . . . . . . . . 19.1.3. Substrates of gluconeogenesis . . . . . . . . . . . . . . . 19.1.4. Energy balance . . . . . . . . . . . . . . . . . . . . . . . 19.1.5. Regulation of gluconeogenesis . . . . . . . . . . . . . . . 19.2. Glycogen Metabolism . . . . . . . . . . . . . . . . . . . . . . . 19.2.1. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2.2. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2.3. Degradation . . . . . . . . . . . . . . . . . . . . . . . . . 19.2.4. Difference between liver and muscle . . . . . . . . . . . . 19.2.5. Regulation of glycogen metabolism . . . . . . . . . . . . 19.2.6. Glycogen storage diseases . . . . . . . . . . . . . . . . . 19.3. Dietary Fructose and Galactose . . . . . . . . . . . . . . . . . . 19.3.1. Fructose metabolism . . . . . . . . . . . . . . . . . . . . 19.3.2. Galactose metabolism . . . . . . . . . . . . . . . . . . . 19.4. The Pentose Phosphate Pathway . . . . . . . . . . . . . . . . . 19.4.1. Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4.2. Products and regulation . . . . . . . . . . . . . . . . . . 19.4.3. Physiological role . . . . . . . . . . . . . . . . . . . . . . 19.4.4. Glucose-6-phosphate dehydrogenase deficiency . . . . . . 19.5. The ‘Minor’ Pathways . . . . . . . . . . . . . . . . . . . . . . . 19.5.1. The Polyol Pathway . . . . . . . . . . . . . . . . . . . . 19.5.2. Synthesis of Amino Sugars . . . . . . . . . . . . . . . . . 19.5.3. The uronic acid pathway. . . . . . . . . . . . . . . . . . 19.6. Practice Questions . . . . . . . . . . . . . . . . . . . . . . . . . 19.7. Objectives in Summary . . . . . . . . . . . . . . . . . . . . . . 20.Lipid Metabolism 20.1. Structures . . . . . . . . . . . . . . . . 20.2. Utilization of Dietary Fat . . . . . . . 20.3. Adipose tissue . . . . . . . . . . . . . . 20.3.1. Triglyceride . . . . . . . . . . . 20.4. Fatty Acid Oxidation . . . . . . . . . . 20.5. Ketogenesis . . . . . . . . . . . . . . . 20.6. Aberrations Of Fatty Acid Metabolism 20.7. From Carbohydrate To Fat . . . . . . 20.8. Phosphoglyceride Metabolism . . . . .

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349 349 349 349 350 350 350 351 351 351 352 352 352 353 354 354 355 355 355 356 356 357 358 358 358 358 358 359

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20.9. Sphingolipid Metabolism . . . . . 20.10.Cholesterol And Bile Acids . . . 20.11.Lipoprotein Composition . . . . . 20.12.Lipoprotein Metabolism . . . . . 20.13.Lipoproteins and Atherosclerosis 20.14.Lipoprotein Disorders . . . . . . 20.15.Practice Questions . . . . . . . . 20.16.Objectives In Summary . . . . .

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369 370 371 373 374 375 376 377

V. Semester two, Mini II

379

21.Nutritional Management of Disease 21.1. Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2. Weight Reduction . . . . . . . . . . . . . . . . . . . . . . . 21.3. Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . 21.3.1. Types of diabetes mellitus . . . . . . . . . . . . . . 21.3.2. Management of Diabetes . . . . . . . . . . . . . . . 21.4. Coronary Heart Disease (CHD) . . . . . . . . . . . . . . . 21.4.1. Dietary Guidelines for Reducing Blood Cholesterol 21.5. Prevention of Coronary Heart Disease (CHD) . . . . . . . 21.6. Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . 21.7. The Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.7.1. Disease of the Biliary System . . . . . . . . . . . . 21.7.2. Parenchymal Liver Disease . . . . . . . . . . . . . 21.8. The Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . 21.8.1. Glomerulonephritis and Nephrotic Syndrome . . . 21.8.2. End-Stage Renal Disease . . . . . . . . . . . . . . . 21.9. Nutritional Therapy In Surgery And Injury . . . . . . . .

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381 381 382 383 383 385 387 388 391 393 396 397 398 399 400 401 402

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405 405 406 408 408 410 414 415 418 418 422 422

22.Amino Acid Metabolism 22.1. Protein metabolism . . . . . . . . . . . . . . . . . . . . . . 22.1.1. Biological value of proteins . . . . . . . . . . . . . 22.2. Nitrogen metabolism . . . . . . . . . . . . . . . . . . . . . 22.2.1. Nitrogen transfer . . . . . . . . . . . . . . . . . . . 22.2.2. Urea-cycle . . . . . . . . . . . . . . . . . . . . . . . 22.3. Catabolism of the carbon backbone . . . . . . . . . . . . . 22.3.1. Ala and Ser enter glycolysis . . . . . . . . . . . . . 22.3.2. Glu, Gln, Asp and Asn enter the Krebs-cycle . . . 22.3.3. Gly, Thr and Ser . . . . . . . . . . . . . . . . . . . 22.3.4. The sulphur-containing amino acids: Met and Cys 22.3.5. Branched-chain amino acids: Val, Ile, Leu . . . . .

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22.3.6. Phe and Tyr . . . . . . . . . . . . . . . . . . 22.3.7. Trp and Lys . . . . . . . . . . . . . . . . . . . 22.3.8. Pro, Arg, His and ornithine . . . . . . . . . . 22.4. Compounds derived from amino acids . . . . . . . . 22.4.1. Carnitine . . . . . . . . . . . . . . . . . . . . 22.4.2. Creatine and creatinine . . . . . . . . . . . . 22.4.3. Polyamines . . . . . . . . . . . . . . . . . . . 22.5. Physiology of amino acids . . . . . . . . . . . . . . . 22.5.1. Metabolism of amino acids . . . . . . . . . . 22.5.2. Inherited amino acid transporter deficiencies . 22.6. Exercises . . . . . . . . . . . . . . . . . . . . . . . . . 22.7. Objectives in Summary: Amino acid metabolism . . 23.Biochemistry of Digestion 23.1. Digestion . . . . . . . . . . . . . . . . 23.1.1. Digestive secretions in man . . 23.1.2. Undigestible materials . . . . . 23.1.3. Lactose intolerance . . . . . . . 23.1.4. Zymogens . . . . . . . . . . . . 23.2. Intermediary metabolism . . . . . . . . 23.3. Carbohydrate metabolism . . . . . . . 23.4. Fat metabolism . . . . . . . . . . . . . 23.5. Metabolism of amino acids and protein 23.6. Alcohol Metabolism . . . . . . . . . . 23.7. Intermediary metabolism . . . . . . . . 23.8. Questions . . . . . . . . . . . . . . . . 23.9. Digestion: Objectives in summary . . .

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24.Heme, Purines and Pyrimidines 24.1. Heme Biosynthesis . . . . . . . . . . . . . . . . . 24.1.1. Disorders of Heme Biosynthesis . . . . . . 24.2. Heme Degradation . . . . . . . . . . . . . . . . . 24.2.1. Hyperbilirubinemia and Jaundice . . . . . 24.3. Purines . . . . . . . . . . . . . . . . . . . . . . . 24.3.1. Purine Biosynthesis . . . . . . . . . . . . 24.3.2. Purine Degradation . . . . . . . . . . . . . 24.4. Pyrimidine Metabolism . . . . . . . . . . . . . . 24.5. Salvage pathways . . . . . . . . . . . . . . . . . . 24.6. Deoxyribonucleotides . . . . . . . . . . . . . . . . 24.7. Anti-neoplastic and anti-bacterial drugs acting on 24.8. Hyperuricemia and Gout . . . . . . . . . . . . . . 24.9. Objectives in Summary . . . . . . . . . . . . . .

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425 427 429 431 431 432 432 435 435 435 436 437

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439 439 439 445 445 447 447 449 450 452 452 454 455 455

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457 457 457 459 461 463 463 464 464 467 467 468 469 471

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Contents

25.Nutrition During the Life Cycle 25.1. Nutrition in Pregnancy and Lactation . . . . 25.2. Breastfeeding . . . . . . . . . . . . . . . . . . 25.2.1. Composition of Human Milk . . . . . 25.2.2. Dietary recommendations for lactating 25.3. Feeding the Weaning Age Group . . . . . . . 25.4. Feeding the School Child . . . . . . . . . . . . 25.5. Feeding Adolescents . . . . . . . . . . . . . . 25.6. Nutrition in the Elderly . . . . . . . . . . . . 25.7. Objectives in Summary . . . . . . . . . . . . 26.Cell Cycle Control and Cancer 26.1. Cell Cycle Control . . . . . . . . . . . . 26.2. Normal Cells in Culture . . . . . . . . . 26.3. Cyclins and the Retinoblastoma Protein 26.4. p53 and the Damage Response . . . . . 26.5. Growth Control by External Stimuli . . 26.6. Mitogenic Signaling . . . . . . . . . . . . 26.7. Principles of Malignant Transformation . 26.8. Cellular Oncogenes . . . . . . . . . . . . 26.9. Nuclear Proteins in Cancer . . . . . . . 26.10.Virally-Induced Cancers . . . . . . . . . 26.11.Inherited Cancer Susceptibility . . . . . 26.12.Objectives in Brief . . . . . . . . . . . .

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485 485 486 486 487 488 489 490 491 492 493 493 495

27.Immunoglobulins and Immunogenetics 27.1. Antibody Structure . . . . . . . . . . . . . . . . . . . 27.1.1. Structure of Immunoglobulin G (IgG) . . . . 27.1.2. Heterogeneity of Immunoglobulins . . . . . . 27.1.3. Other Ig Domain Proteins . . . . . . . . . . . 27.2. Immunogenetics . . . . . . . . . . . . . . . . . . . . . 27.2.1. The Major Histocompatibility Locus (MHC) 27.2.2. Immunoglobulin Gene Structure . . . . . . . 27.3. MHC (HLA) and Clinical Risk of Disease . . . . . . 27.3.1. MHC Polymorphisms and Disease Risk . . . . 27.3.2. Familial Immune Disorders . . . . . . . . . . 27.4. Objectives in Brief . . . . . . . . . . . . . . . . . . .

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497 497 497 499 500 501 501 506 508 508 508 509

28.Inherited diseases of metabolism 28.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.1.1. Significance of inherited diseases of metabolism . . . . . . . . . . . . 28.1.2. Mechanism of IDoM . . . . . . . . . . . . . . . . . . . . . . . . . . .

511 511 511 511

xiv

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Contents

28.1.3. Newborn screening . . . . . . . . . . . . . . 28.2. Cytosolic enzymes . . . . . . . . . . . . . . . . . . 28.2.1. G6PDH deficiency – Favism . . . . . . . . . 28.2.2. Galactosemia . . . . . . . . . . . . . . . . . 28.2.3. Errors of fructose metabolism . . . . . . . . 28.2.4. Lactase persistence/restriction . . . . . . . 28.2.5. Amino acids . . . . . . . . . . . . . . . . . . 28.3. Glycogen storage diseases . . . . . . . . . . . . . . 28.4. Lysosomes . . . . . . . . . . . . . . . . . . . . . . . 28.4.1. I-cell disease . . . . . . . . . . . . . . . . . . 28.4.2. Mucopolysaccharidoses and sphingolipidoses 28.5. Mitochondria . . . . . . . . . . . . . . . . . . . . . 28.5.1. Pyruvate metabolism . . . . . . . . . . . . . 28.5.2. β-oxidation of fatty acids . . . . . . . . . . 28.6. Peroxisomes . . . . . . . . . . . . . . . . . . . . . . 28.7. Endoplasmic reticulum . . . . . . . . . . . . . . . . 28.7.1. Biotransformation . . . . . . . . . . . . . . 28.8. Objectives . . . . . . . . . . . . . . . . . . . . . . .

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513 516 516 517 518 518 519 519 520 521 522 528 528 532 533 535 535 543

VI. Semester two, Mini III

545

29.Advanced DNA technology 29.1. Germline Gene Manipulations (analysis of gene function) 29.1.1. Cre/LoxP system for recombination . . . . . . . 29.2. RNAi (inhibitory RNA, Knock-down) . . . . . . . . . . 29.3. Microarray Technology (DNA Chips) . . . . . . . . . . . 29.4. Somatic Gene Therapy . . . . . . . . . . . . . . . . . . . 29.5. Proteomics Methods . . . . . . . . . . . . . . . . . . . . 29.6. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . .

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547 547 549 550 550 551 553 553

30.Population Genetics and Genetic Counseling 30.1. Genotype Frequencies . . . . . . . . . . . 30.1.1. The Hardy-Weinberg equation . 30.2. Inbreeding . . . . . . . . . . . . . . . . . . 30.3. Mutation and Selection . . . . . . . . . . . 30.4. Genetic Counseling . . . . . . . . . . . . . 30.5. Diagnostic Strategies . . . . . . . . . . . . 30.6. Prenatal Diagnosis . . . . . . . . . . . . . 30.7. Aim . . . . . . . . . . . . . . . . . . . . . 30.8. Screening Tests . . . . . . . . . . . . . . . 30.9. Assisted Reproduction . . . . . . . . . . .

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555 555 555 557 557 560 561 562 562 563 564

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xv

Contents

30.10.Objectives in Brief . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567 31.Integration of Metabolism 31.1. Regulation of Enzyme Activity . . . . . . . . . 31.1.1. Hormonal Control . . . . . . . . . . . . 31.1.2. Metabolic role of organs . . . . . . . . . 31.2. The respiratory quotient . . . . . . . . . . . . . 31.3. The Starve-Feed Cycle . . . . . . . . . . . . . . 31.3.1. Phases of the starve-feed cycle . . . . . 31.3.2. Role of organs during starve-feed cycles 31.3.3. Unbalanced meals . . . . . . . . . . . . 31.4. Obesity . . . . . . . . . . . . . . . . . . . . . . 31.4.1. Adipose tissue in obesity . . . . . . . . . 31.4.2. Appetite control . . . . . . . . . . . . . 31.4.3. Role of gut flora . . . . . . . . . . . . . 31.4.4. Beneficial effects of dietary restriction . 31.4.5. Epigenetics . . . . . . . . . . . . . . . . 31.4.6. Systems biology . . . . . . . . . . . . . . 31.4.7. Metabolic syndrome . . . . . . . . . . . 31.5. Diabetes . . . . . . . . . . . . . . . . . . . . . . 31.6. Example questions . . . . . . . . . . . . . . . . 31.7. Objectives in Summary . . . . . . . . . . . . .

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569 569 569 570 571 574 574 576 583 583 584 586 586 589 589 594 594 595 604 605

32.Multifactorial Inheritance 32.1. Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . 32.2. Quantitative Traits . . . . . . . . . . . . . . . . . . . . . 32.3. Common Diseases . . . . . . . . . . . . . . . . . . . . . . 32.4. Congenital Malformations (“Birth Defects”) . . . . . . . 32.4.1. Nongenetic Causes Of Congenital Malformations 32.5. Quantitative Trait Loci (QTLs) . . . . . . . . . . . . . . 32.6. Examples Of Susceptibility Genes . . . . . . . . . . . . . 32.7. Objectives in Brief . . . . . . . . . . . . . . . . . . . . .

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607 607 608 610 611 612 615 616 617

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VII. Appendix 33.Answers to the example questions 33.1. Thermodynamics . . . . . . . . 33.2. Proteins . . . . . . . . . . . . . 33.3. Enzymes . . . . . . . . . . . . . 33.4. Amino acid metabolism . . . . 33.5. Digestion . . . . . . . . . . . .

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621 621 623 625 626 627

Contents

33.6. Integration of metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628 34.Tables 34.1. Conversion from non-metric to metric units 34.2. Symbols used . . . . . . . . . . . . . . . . . 34.3. Greek alphabet . . . . . . . . . . . . . . . . 34.4. The genetic code . . . . . . . . . . . . . . . 34.5. Periodic system of the elements . . . . . . . 35.Acronyms

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629 630 630 631 632 634 635

xvii

Welcome The faculty of the Biochemistry Department at Ross University welcome you to the biochemistry course. The following handouts are provided to supplement lectures and reading material in your text to complement the required textbooks: G. Meisenberg & W.H. Simmons: Principles of Medical Biochemistry, 2nd ed., Philadelphia (Mosby) 2006 L.B. Jorde, J.C. Carey, M.J. Bamshad & R.L. White: Medical Genetics, 3rd updated ed., Philadelphia (Mosby) 2006 You should look at the appropriate chapters of both the handout and your textbooks before a lecture, this will enhance your learning success. Please understand that this handout set is in no way a sure indication of what you will be required to know for exams. This material can be viewed as the minimum information students are expected to know. Additional reading material for this course can be found in the library. Reserve materials, texts and journals in this subject area are also available. We encourage you to use these materials in addition to your assigned materials throughout the semester. Enhancing and updating your knowledge of biochemistry requires you to take the long view, and develop habits which foster lifetime learning. Discussion of medical biochemistry topics with faculty and other students is helpful for integrating this core knowledge with your other subjects. The following web-sites are a small sampling of additional material which can be accessed online. We encourage your use of internet resources to enhance your integration of course material. General Biochemistry Courses: http://www.kumc.edu/biochemistry/bioc800/biocindx.htm http://web.indstate.edu/thcme/mwking/ http://tutor.lcsf.ucsb.edu/instdev/sears/biochemistry/ Metabolism: http://www.gwu.edu/ mpb/ Clinical Biochemistry: http://www.qub.ac.uk/cm/cb/text/studgide/index.htm

xix

Biochemistry and Genetics

Database searching for journal articles, and biochemical information: http://www3.ncbi.nlm.nih.gov/Entrez/index.html Enjoy your learning experience in this course! And remember, the faculty are here to help you succeed. Sincerely yours, The Biochemistry Faculty

xx

Part I.

Semester one, Mini I

1. Introduction to Biomolecules 1.1. Elements and molecules Biomolecules contain only a few of the 92 elements that exist in nature: carbon hydrogen, oxygen (all), nitrogen (proteins, nucleic acids, vitamins), sulfur (proteins, vitamins, intermediates), phosphate (nucleic acids, some proteins, many metabolic intermediates). Some elements occur in charged form: sodium, potassium, calcium, magnesium, chloride. If you know the composition of a molecule, you know its molecular weight. Example: Glucose (C6 H12 O6 ) has a molecular weight (MW) of 180. One mol is the molecular weight in grams (g). 1 mol of glucose is 180 g. Concentrations of dissolved molecules are often given in mol per liter (mol/L, or M), millimole per liter (mmol/L), or milligrams per 100 ml (90 mg/dl). Can you calculate its molar concentration?

1.2. Covalent Bonds And Non-covalent Interactions 1.2.1. Covalent bonds The covalent bonds that connect the atoms in molecules are formed by binding electron pairs which orbit both atoms. Formation and breakage of covalent bonds requires chemical reactions which, in the body, are catalyzed by enzymes.

Element Carbon (C) Hydrogen (H) Oxygen (O) Nitrogen (N) Sulfur (S) Phosphorus (P)

Table 1.1.: Important elements biol. abund. terrest. abund. atomic mass (% w/w) (% w/w) (Da) 18.5 0.030 12 9.5 0.140 1 65.0 65.0 16 3.3 0.005 14 0.3 0.050 32 0.2 0.120 31

3

1.2.2

Biochemistry and Genetics

Table 1.2.: Composition of the human body Class of Molecule Content (%) MW (Da) Water 60.0 18 Inorganic salt, soluble 0.7 = atomic weight Inorganic salt, insoluble 5.5 = atomic weight Protein 16.0 5 × 103 –1 × 106 Triglyceride (fat) 13.0 ≈ 800 Membrane lipids 2.5 400–1000 Carbohydrates 1.5 >1000 (polysaccharides) Nucleic acids 1.2 2 × 104 –2 × 108 Electronegativity is the tendency of an atom to attract electrons. The order of electronegativities of atoms in biomolecules is: O>N>S>C≈H

1.2.2. Non-covalent interactions The binding electron pair of a covalent bond is displaced towards the more electronegative atom. This creates a dipole, with partial positive and negative charges at opposite ends. Opposite charges attract each other, therefore dipole-dipole interactions can form between different polarized bonds. Hydrogen bonds are a type of dipole-dipole interaction involving a hydrogen bound to an electronegative atom (O or N). The water molecule is a dipole with a partial negative charge on the oxygen and partial positive charge on the hydrogens: The unusual physical properties of water (for example its high boiling point) are caused by hydrogen bonds between water molecules. δ+ H

O

δ+ H

δ-

Ions carry either a positive charge (cations) or a negative charge (anions). They form iondipole interactions with water. That is why many salts are water soluble: the interactions with water can overcome the electrostatic interaction (“salt bond”) in the salt crystal. Organic molecules that contain positive and negative charges interact strongly with water, and most of them are water soluble. Also molecules that form hydrogen bonds with water are water soluble. Molecules that have mostly nonpolar bonds, (especially the hydrocarbons which consist of carbon and hydrogen) are insoluble in water.

4

1.3

Bonds in biomolecules H2N

NH2

C

H2N

>

O

C

CH3

O

H3C

>

CH3

O Acetone

Acetamide

Urea

C

H3C

>

C H2

CH3

Propane

Polar interactions (ion-ion, ion-dipole, dipole-dipole) are also important for interactions within and between biomolecules. Hydrophobic interactions are formed between nonpolar groups (usually hydrocarbon) in the molecules. Nonpolar groups occupy space between the water molecules preventing their interactions. Therefore, hydrophobic groups aggregate to minimize their interface with water. Van-derWaals interactions are weak non-covalent forces between neighboring molecules. All noncovalent bonds are weak. They form and break spontaneously, therefore all non-covalent bonding is reversible.

1.3. Bonds in biomolecules Functional groups determine chemical reactivity and physical properties (solubility, melting point, boiling point) of the molecule. Most important functional groups (R= “residue”, the remainder of the molecule):

Alkyl groups R−CH3 (methyl), R−CH2−CH3 (ethyl) etc. Nonpolar (hydrophobic) groups. Most common in lipids.

Hydroxy group R−OH Non-ionizable, forms dipole-dipole interactions. Occurs in carbohydrates, ethanol, some amino acids.

Carboxy group This is the most important acidic group in biomolecules. In fatty acids, amino acids, and many metabolic intermediates. The carboxy group carries a negative charge at pH = 7. O

C R

O

O

_ C R

O

O

C

O

R

5

1.3

Biochemistry and Genetics

Carbonyl group Non-ionizable, forms dipole-dipole interactions. Occurs in monosaccharides. O

C

O

H

C

R

R

R

aldehyde

ketone

Amino group These are the most important basic groups in biomolecules. In amino acids and biogenic amines. The nitrogen of aliphatic amines carries a positive charge at pH = 7. H

H

R"

R N|

R N|

R N|

H

R'

R'

secondary amine

primary amine

R" +

R N R'" R'

tertiary amine

quarternary ammonium base

Sulphydryl group R−SH Weakly acidic. In cysteine, and coenzyme A. Hemiacetal Non-ionizable, in the ring forms of monosaccharides. OH

R C H OR' Hemiacetal

Most biomolecules are large molecules (macromolecules) which are formed when functional groups in their building blocks react with the formation of water. These reactions are called condensation reactions. The reversal of a condensation reaction is called hydrolysis. Condensation reactions are endergonic (energy-requiring), while hydrolysis reactions are exergonic (energy-releasing): Carboxylic ester From carboxy group and hydroxy group. Example: Triglycerides (fat) O R C

O

+

HO R"

R C O R"

OH

+

H-O-H

Phosphate ester From hydroxy group and phosphate. Example: Many intermediates of carbohydrate metabolism. All esters can be cleaved by acid and, especially, alkaline hydrolysis. O

O P OH O

6

O

+

HO R

O P O R O

+

H-O-H

1.3

Bonds in biomolecules

Phosphodiester From hydroxy group and phosphate ester. Examples: Nucleic acids, phospholipids. O R

O

+

O P OH

R

HO R'

O P

O

O

R'

+

H-O-H

O

Mixed anhydride From two different acids (e.g., carboxylic acid and inorganic phosphate). In some metabolic intermediates. O

O P OH

+

O

O HO

O

O

O P O C R

C R

+

H-O-H

O

Phosphoanhydride From phosphate and phosphate (-ester). Example: Energy-rich bonds in ATP. O

O P OH

+

O P O

HO P O R

O

O

O

O

+

H-O-H

O

O

O

P O R

Ether From two hydroxy groups. In some O-methylated and hydroxy group. R OH

+

HO R'

R O R'

+

H-O-H

Acetal From hemiacetal and hydroxy group. In Carbohydrate, where they are known as glycosidic bonds. R'

R"

O

R'

C OH

+

HO R

R"

H

O

C O R

+

H-O-H

H

Thioesters From sulfhydryl group and carboxy group. In coenzyme A thioesters (acetylCoA, fatty acyl-CoA). R C

O OH

O

+

HS

R'

R C S R'

+

H-O-H

Amides From (carboxylic) acid and ammonia or amine. Example: Peptide bonds in proteins.

7

1.4

Biochemistry and Genetics O

O

+

R C

R C

NH3

OH

NH2

O

+

H-O-H

+

H-O-H

O

+

R C OH

R C

H2N R'

N R' H

The formation of anhydride bonds and thioester bonds requires more energy than the formation of the other bonds. These bonds are called energy-rich bonds.

1.4. Isomers Isomers are chemically different molecules of identical composition. There are three different types: Positional isomers differ in the positions of atoms or functional groups within the molecule. H H

C

O

H C OH C O

H C OH H C OH

H C OH H

H Glyceraldehyde

Dihydroxyacetone

Geometric isomers differ in the relative geometric position of different parts of the molecule, for example cis-trans isomers at double bonds. Substituents at double bonds are planar, and they don’t rotate: R'

H

R'

H

R

H

H

R

cis-isomer

trans-isomer

Optical isomers arise when a carbon has four different substituents making it an asymmetric carbon. If the molecule has only one asymmetric carbon, the isomers are mirror images or enantiomers. Also disastereomers differ only in the position of some groups in space, but they are not mirror images. Unlike positional isomers and diastereomers, enantiomers have identical physicochemical properties. But they rotate the plane of

8

1.5

Acids and bases

polarized light in opposite directions. Optical isomerism is also called chirality. Example: O

C

O

O +

H C N H3

H3N

+

C

O

C H

R

R

D-amino acid

L-amino acid

Alternative positional, geometric and optical isomers are not equivalent biologically. Enzymes can distinguish between the isomers.

1.5. Acids and bases A proton can be transferred from one water molecule to another in a spontaneous, reversible reaction 2H2 O * ) H3 O+ + OH− In distilled water, [H3 O+ ] = [OH− ] = 1 × 10−7 M, and [H3 O+ ] × [OH− ] = 10 × 10−14 M2 . This means that any rise in [H3 O+ ] must be compensated by a decline in [OH− ] and vice versa. The concentration of [H3 O+ ] is usually written as the “proton concentration” [H+ ], although free protons are uncommon in water. The proton concentration is expressed as the pH value. The pH value is the negative logarithm of [H+ ]. pH = 7 neutral ([H+ ] = 1 × 10−7 M) pH < 7 acidic ([H+ ] > 1 × 10−7 M) pH > 7 alkaline ([H+ ] < 1 × 10−7 M) The pH of cells and body fluids has to be kept constant. Normal blood pH: 7.4. Acids are substances which donate protons to water. They increase [H+ ] and decrease the pH. Bases are substances which accept protons from water: They decrease [H+ ] and increase the pH. Many biomolecules contain acidic and basic groups which undergo reversible protonation/deprotonation reactions. The protonation state of such groups depends on the solvent pH. This can be understood in terms of mass action: H+ is a reactant, therefore its concentration drives protonation and deprotonation reactions. The major acidic group in biomolecules is the carboxy group, which undergoes the following ionization reaction: R C

O

OH

+

H2O

R C

O O

+

H3O+

9

1.6

Biochemistry and Genetics

This is a nonenzymatic, instantaneous, freely reversible equilibrium reaction. Note that the carboxylate anion (R − COO− ) is itself a base, ready to accept a proton and reform the carboxylic acid in the reverse reaction. The carboxylate anion is the conjugate base of the carboxylic acid. Acids are uncharged in the protonated form and negatively charged (anionic) in the deprotonated form. The major basic group in biomolecules is the primary amino group:

+

R NH2

+

R N H3

H3O+

+

H2O

The ammonium salt (R − N+ H3 ) is itself an acid, ready to release a proton and reform the primary amino group in the reverse reaction. The ammonium salt is the conjugate acid of the amine. Bases are positively charged (cationic) in the protonated form and uncharged in the deprotonated form. The protonation/deprotonation reaction R C

O OH

+

H2O

R C

O O

+

H3O+

can be described by its dissociation constant Ka : Ka =

[R−COO− ] × [H + ] [R−COOH]

or

[H + ] = Ka ×

[R−COOH] [R−COO− ]

(1.1)

We can put this whole equation into the negative logarithm: pH = pK a − log(

[R−COO− ] [R−COOH] ) = pK + log( ) a [R−COO− ] [R−COOH]

(1.2)

The pKa value is an intrinsic property of the ionizable group. If the pH equals the pKa , the group is half-protonated; if pH > pKa it is mostly deprotonated; if pH < pKa , it is mostly protonated. Important ionizable groups in biomolecules: Acidic groups, deprotonated at pH 7: Carboxy group, phosphate ester, phosphodiester. Acidic groups, protonated at pH 7: Sulfhydryl group, phenolic hydroxy group. Basic groups, protonated at pH 7: Aliphatic amino groups. Basic groups, deprotonated at pH 7: Aromatic amines.

10

1.6

Fats and carbohydrates

1.6. Fats and carbohydrates Triglycerides (“fat”) are esters formed from glycerol and fatty acids H2C OH HO CH C OH H2

Glycerol

H2 C

H3C

C H2

H2 C

C H2

H2 C

H2 C

C H2

C H2

H2 C

C H2

H2 C

H2 C

C H2

C H2

COOH

Fatty acid (Palmitic acid)

O H O

O H 2 H2C C C

Palmitic acid is a saturated fatty acid. Monounsaturated fatty acids have a single C=C double bond, and polyunsaturated fatty acids have more than one C=C double bond.

Triglycerides are not water-soluble. They are used as energy stores in adipose tissue. Nonpolar molecules like the triglycerides are called lipids. Lipids other than triglycerides occur in biological membranes. They include the phospholipids, glycolipids and cholesterol. Carbohydrates are made from monosaccharides. These are polyalcohols containing an aldehyde group (aldoses) or keto group (ketoses). Monosaccharides may have: 3 carbons Trioses 6 carbons Hexoses 4 carbons Tetroses 7 carbons Heptoses 5 carbons Pentoses etc. Examples: O

CH

HC OH HO CH

O

CH

HC OH HO CH

O

CH

CH2OH

O

CH

HO CH

O C

HC OH

HO CH

HO CH

HC OH

HC OH

HC OH

HC OH

HC OH

HC OH

HC OH

HC OH

HC OH

CH2OH

CH2OH

CH2OH

CH2OH

CH2OH

HO CH

D-Galactose

Gal

D-Glucose

Glu

D-Mannose

Man

D-Fructose

Fru

D-Ribose

Rib

Only D-isomers are shown as these are prevalent in nature. Gal, Glc and Man are not enantiomers but diastereomers because they are not mirror

11

1.7.1

Biochemistry and Genetics

images. Monosaccharides that are distinguished by the orientation of substituents around a single asymmetric carbon are called epimeres.

Most monosaccharides form ring structures by a hemiacetal or hemiketal bond between the carbonyl group and one of the hydroxy groups: CH2OH

O

O HO OH HO

OH

OH OH

β-D-glucose

OH

OH CH2OH OH

O

α-D-glucose

OH

D-glucose

OH

OH OH

Glucose spends 99.9975 % of its time in the ring form. C-1 in the ring form is asymmetric. It gives rise to α and β isomers which are called anomers. Because the ring can open and close spontaneously, α and β equilibrate until 34 % is α and 66 % is β. This is called mutarotation.

Disaccharides (from 2 monosaccharides), (from “a few” monosaccharides) and (from “many” monosaccharides) are formed by . These are acetal or ketal bonds that involve at least one anomeric (aldehyde or keto carbon). Glycosidic bonds involving the anomeric carbon of a carbohydrate can also be formed with non-carbohydrate components. Important disaccharides: Maltose Glucose+ Glucose (α-1,4 bond) Lactose Galactose + Glucose (β-1,4 bond) Sucrose Glucose + Fructose (α, β-1,2 bond) Important polysaccharides Starch Glucose, with α-1,4 bonds and some α-1,6 bonds. Glycogen Like starch, but with more α-1,6 bonds Cellulose Glucose, with β-1,4 bonds Monosaccharides and disaccharides are water soluble. Polysaccharides are hydrated, but not all are water soluble. Monosaccharides have reducing properties because of the presence of the carbonyl carbon.

12

Molecular Structure

1.7.1

1.7. Objectives in summary 1.7.1. Molecular Structure 1. Recognize in chemical formulas the important functional groups and bond types, including hydroxy, carbonyl, carboxy, sulfhydryl and amino groups, and ester, ether, glycosidic, thioester and amide bonds. 2. Know the relative electronegativities of O,N,S,C and H. State that chemical bond formation is usually endergonic while hydrolytic bond cleavage is exergonic. 3. Define the term “energy rich bond”. 4. Describe the principal types of non-covalent interaction between biomolecules, including hydrogen bonds, salt bonds, van der Waals interactions and hydrophobic interactions. 5. Describe structural features of biomolecules, which tend to increase or decrease water solubility, including the effect of pH and charge pattern. 6. Know the definition of terms, such as, redox reaction, anion, cation, zwitterions, pH value, isoelectric point and buffering capacity. 7. Application of the Henderson-Hasselbalch equation to problems in which either the pH the pKa or the ratio of protonated/unprotonated form has to be calculated. 8. Recognition of asymmetric carbons in chemical structures and predict existence of optical isomers. 9. Recognize the general structures of glycerol, fatty acids, and triglycerides in the chemical formula. 10. Describe the properties of fatty acids and triglycerides with respect to water solubility, chemical stability of the ester bond and effects of pH changes on protonation state water solubility. 11. Recognition of the general structure of monosaccharides both in open -chain form and the Hayworth projection. 12. Define the terms aldose, ketose, triose, pentose, hexose, hemiacetal, hemiketal, and glycosidic bond. 13. Name the chemical building blocks and bond types in nucleic acids.

13

2. Energy changes and rates of chemical reactions Thermodynamic properties are those features of a chemical reaction that are related to its energy balance and equilibrium. Kinetic properties are those related to the rate (velocity) of the reaction. Only the kinetic properties of reactions are changed by enzymes.

2.1. Thermodynamics The following definitions of important terms are oversimplified, but sufficient for the purpose of medical biochemistry. It is important that you know them as they will be frequently used to describe reactions that go on in our body. Temperature is the random movement of molecules, and is measured in K. 0 K is the lowest theoretically possible temperature, where not only all molecules, but even the electrons of their atoms, would be at rest. In practice, this temperature can never be reached. Note that temperatures are measured in Kelvin (K), but temperature differences in °. There is no such thing as °K. Alternatively, temperatures may also be measured in degrees Celsius °C (confusing, isn’t it?). You convert from °C to K by adding 273.16. In formulas, the capital T represents temperature. In the US the unit °F was used in the past, but you should avoid any non-SI units, they make a lot of additional work and are a source of calculation error. Heat (q) flows between bodies of different temperature, with q = C × ∆T . C is the heat capacity of the bodies (amount of energy you need to increase the temperature by a given amount), and ∆T the difference in temperature between them. The Greek letter ∆ (Delta) always symbolizes differences. In formulas, the heat leaving a body is denoted with a negative, heat entering a body with a positive sign. For an example see fig. 2.1. Heat is measured in Joule J. Work w is anything that “somehow” can be converted to the lifting of weights. When work is transferred across system boundaries then work done on a system gets a positive, system done by a system a negative sign. The unit for work is the Joule (J). Energy is the sum of heat and work: E = q + w = const

15

(2.1)

2.1

Biochemistry and Genetics

Figure 2.1.: Heat flowing between bodies of different temperatures. For explanations see text.

40 °C

60 °C

0 °C

20 °C

30 °C

30 °C

In other words, energy can not be created nor destroyed. This statement is called the first law of thermodynamics. Since both heat and work are measured in J, energy of course has that unit too. A system is some part of the universe, which is separated from the rest (see fig. 2.2). We distinguish: Open systems can exchange both matter and energy with the rest of the environment. Example: an open test tube. Closed systems can exchange energy, but not matter with the environment. A stoppered test tube would be an example, matter can no longer leave or enter, but we may still heat its content. Insulated systems (also called “adiabatic” systems) can exchange neither matter nor energy with the environment. If we placed our stoppered test tube into a Dewar-vessel, energy exchange would be prevented. Such vessels have a double-layer construction, with vacuum as insulator between the inner and outer glass bottle to minimize conduction and convection. The bottles have a silver coating on the vacuum side, to limit radiation. You may have used such vessels to keep your coffee hot. Standard conditions: Some thermodynamic properties of a system depend on the conditions that system is in. Chemists have agreed to reference their measurement to a pressure of 1014 hPa (standard atmospheric pressure), a temperature of 25 °C (= 298 K) and a concentration of 1 M for all reactants. The latter point is an inconvenience for biochemists, because if [H+ ] = 1 M, then pH= 0 (see later). Very few biochemical reactions occur at such low a

16

2.1

Thermodynamics

Figure 2.2.: From the left to the right: open, closed and insulated (adiabatic) systems.

open

closed

insulated

pH, so biochemical standard conditions are pH 7. Biochemists also define standard values for ionic strength (250 mM) and [Mg2+ ] (3 mM), since these influence the activity of many enzymes. The values chosen simply reflect those that we find in living cells. If a parameter X is measured under chemical standard conditions, we write X 0 , if it was measured under biological standard conditions we write X 00 . In older books you may find X 0 ’ instead, because IUPAC changed the nomenclature several years ago. Enthalpy (H) is the heat of a chemical reaction. It is expressed in Joule/mol (J/mol). Enthalpy itself can not be measured, but changes in enthalpy (∆H) can. To do these measurements, we have to assign the value 0 J/mol to some arbitrary state. By convention, this value is assigned to pure elements in their stable state under standard conditions. If heat is produced by a reaction, we give ∆H a negative sign and call the reaction exothermic. If enthalpy is consumed during a reaction, we give ∆H a positive sign, such reactions are called endothermic. The change in enthalpy is given by: ∆H = ∆E + P × ∆V

(2.2)

where P is the pressure (in Pa) and ∆V the change in volume. The product P × ∆V is the mechanical work done during a reaction. Because biochemical reactions usually happen in dilute aqueous solutions, ∆V ≈ 0 and hence ∆H ≈ ∆E. This is a useful result because it is

17

2.1

Biochemistry and Genetics

difficult to measure a change in energy, but much easier in to measure changes in enthalpy. For biochemical reactions we can thus use ∆H as an approximation for ∆E, which is the much more important parameter. Entropy (S) is a measure of chaos. If you look at fig. 2.1, you see two bodies of different temperature. If you bring them close together, heat could theoretically flow from the cold body to the hot, making the cold body even colder and the hot body even hotter. The first law of thermodynamics would allow that, because the energy lost by the cold body exactly balances the energy gained by the hot. However, everyday experience shows us that this is not what will happen. Instead, heat will flow from the hot body to the cold, until both have the same temperature. This is an example for a general principle called the second law of thermodynamics: The direction of a reaction is the one where the entropy (disorder) of the universe is maximized, that is ∆S ≥ 0. Note: The second law of thermodynamics allows for a local reduction of entropy as long as the global entropy increases. Life does exactly that: Our sun sends out energy in form of photons, constantly increasing universal entropy. Plants catch this orderly stream of photons and dissipate it, again increasing the entropy of the universe. However, part of this entropy difference is not given up to the universe, but saved in the form of organic compounds like sugar, which animals then consume. There is a third law of thermodynamics which states that the entropy of an ideal crystal at a temperature of 0 K is 0 J mol−1 K−1 . This law allows us to measure entropies, but is of no other consequence to biochemistry. You may safely assign it to passive knowledge. Water below 100 °C is liquid, above that temperature it is gaseous, but we have to expend energy to convert water of 100 °C into steam of 100 °C (the heat of evaporation). Gases, of course, have a much higher entropy than liquids. Thus energy, temperature and entropy of a reaction must be linked. This linkage is provided by the Gibbs free energy ∆G through the Gibbs-Helmholtz-equation: ∆G = ∆H − T ∆S

(2.3)

∆G is a very important parameter, which we will encounter again and again in this course. It determines the direction of a chemical reaction: ∆G < 0: reaction proceeds from left to right (v+ > v− ) ∆G = 0: reaction is at equilibrium (v+ = v− ) ∆G > 0: reaction proceeds from right to left (v+ < v− ) By convention, chemical reactions are always written in the direction where ∆G is negative. ∆G is that part of ∆H of a reaction that can be used to do useful work. T ∆S is the part of ∆H which is lost as heat. Therefore ∆G determines the energy efficiency of a reaction.

18

Reaction kinetics

2.2

This is, of course, a key driving force in evolution since any energy not lost as heat may be used for growth and reproduction. Thus it is not surprising that the energy efficiency of living organisms is close to the thermodynamically allowed maximum, and often much higher than that of man-made devices. Physical processes in which chemical bond energies don’t change (∆H = 0, for example diffusion across a membrane) are also driven by ∆G via an increase in entropy (i.e. a negative value for [T × ∆S]). The body has to use chemical bond energy to combat the tendency for increasing entropy. That’s what metabolic energy is good for! Summary: Exothermic reaction: Endothermic reaction: Exergonic reaction: Endergonic reaction: Equilibrium:

∆H < 0, heat is released. ∆H > 0, heat is absorbed ∆G < 0, reaction can proceed ∆G > 0, reaction cannot proceed. ∆G = 0 concentration of reactants doesn’t change over time, as the rates for forward and

2.2. Reaction kinetics In theory, all chemical reactions are reversible. If only non-covalent interactions are involved, as in protonation-deprotonation, antigen-antibody binding and hormone-receptor binding, the reactions are always found at equilibrium concentrations. Chemical reactions in which covalent bonds are formed and broken are also reversible, but there is an energy barrier to the reaction. Therefore the reactions are not always found at equilibrium concentrations. The equilibrium is described by the equilibrium constant (Keq ). For the reaction k+1 GGGGGGGB aA + bB + ... F (2.4) GG zZ + yY + ... k−1 we get: order of forward reaction: a + b + ... order of backward reaction z + y + ... velocity of forward reaction v+ = k+1 × [A]a × [B]b × ... velocity of backward reaction v− = k−1 × [Z]z × [Y ]y × ... equilibrium constant Keq =

[Z]z ×[Y ]y ×... [A]a ×[B]b ×...

=

k+1 k−1

(law of mass action)

19

2.2.1

Biochemistry and Genetics

Although the equilibrium constant is a thermodynamic characteristic, it is related to the rate constants of the forward (k+1 ) and reverse reactions (k−1 ), since at equilibrium the rates of the forward reaction and the reverse reaction are equal: k+1 × [A]a × [B]b × ... = k−1 × [Z]z × [Y ]y × ... k [Z]z × [Y ]y × ... = +1 = Keq a b k−1 [A] × [B] × ...

(2.5) (2.6)

The actual free energy change (∆G) for the reaction is: ∆G = ∆G00 + RT × ln



[Z]z × [Y ]y × ... [A]a × [B]b × ...



(2.7)

with R = universal gas constant (8.314 472(15) J mol−1 K−1 ). Note that here the actual, not the equilibrium concentrations are used. At equilibrium, ∆G = 0, therefore there is a logarithmic relationship between Keq and ∆G00 : Keq ∆G00 (kJ/mol) 1 × 10−5 28.5 1 × 10−4 22.8 1 × 10−3 17.1 1 × 10−2 11.4 1 × 10−1 5.7 1 0.0 1 × 101 -5.7 1 × 102 -11.4 3 1 × 10 -17.1 1 × 104 -22.8 1 × 105 -28.5 Note: By convention, capital K denotes equilibrium constants, small k rate constants. It is important that you do not mix these up!

2.2.1. Order of Reactions Zero-order reaction: The reaction rate v is independent of the substrate concentration. Zero-order reactions are observed only in catalyzed reactions when the catalyst is saturated with substrate. The decrease of the substrate concentration is linear. Formula (k = rate constant, units are mol/s): v=k (2.8)

20

The principle of Le Chatelier

2.3

First-order reaction: The reaction rate is proportional to the substrate concentration (units of k are s−1 ): v = k × [A] (2.9) The decrease of the substrate concentration is asymptotic, and its half-life can be determined.

Second-order reaction: The reaction rate depends on the concentrations of 2 substrates: v = k × [A] × [B]

(2.10)

The unit of k is M−1 s−1 .

Pseudo-first-order reaction: There are 2 substrates, but one is present in large excess and is not rate-limiting. kap describes the apparent rate constant for these conditions. Example: Hydrolysis-reactions: A + H2 O → B + C, the water concentration is 1000 g/l / 18 g/mol = 55.5 mol/l, almost independent of the concentration of the other reactants. Thus the reaction rate will depend only on [A]: v = kap × [A]

(2.11)

2.2.2. The principle of Le Chatelier If the conditions of a reaction are changed, the equilibrium will change in such a way as to counteract the change. This is an extremely important rule, both in chemical technology and in biochemistry. It allows the yield of a chemical reaction to be influenced. Examples: • If you dissolve salts in water, the solution becomes cold. If you increase the temperature, more salt can be dissolved. • If you remove one of the products of a reaction, more reactants are turned into products to maintain the equilibrium constant. Thus by constantly removing the products, you can drive an equilibrium reaction to completion. • If you add one of the reactants in large excess, more of the other reactants is converted to product. This allows you to use an expensive reactant more completely, at the expense of the other (cheaper) ones.

21

2.4

Biochemistry and Genetics

2.3. Catalysis All “true” chemical reactions, catalyzed or uncatalyzed, go through a short-lived unstable transition state (‡): without catalyst

a with catalyst

The transition state has a higher free energy content than the substrate and the product, creating an energy barrier between S and P. This energy barrier, known as the energy of activation (∆Ea ), is the difference in the free energy contents of S and ‡. A condition that is stable kinetically, but is not the most favorable state thermodynamically, is called metastable. The human body is metastable. Catalysts increase the rate of the reaction by decreasing the free energy of activation. The transition state of the catalyzed reaction is different from that of the uncatalyzed reaction. It has a lower free energy content. The equilibrium of the reaction remains unchanged, since only ∆Ea , but not ∆G, is changed. The catalyst is not consumed during the reaction, it may undergo a bond with the substrate temporarily, but is regenerated before the reaction is complete.

2.4. Example questions 1) Entropy, driving force of a reaction The heat of evaporation of water is 40.7 kJ/mol, its boiling point 100 °C. The change in entropy ∆S during evaporation is approximately A 0.109 J mol−1 K−1 B 109 J mol−1 K−1 C 407 J mol−1 K−1 D −109 J mol−1 K−1

22

Example questions

2.4

E −407 J mol−1 K−1 2) Ligand binding to receptor The plasma membrane of a cell contains 10 000 receptors for the hormone X, and 1000 need to have hormone bound to stimulate the maximal response in the cell.The dissociation constant Kd = 1 nM. How high does the concentration of the hormone need to be to get maximal response of the cell? Carefull: there is a trick in this question! A 0.05 nM B 0.11 nM C 0.25 nM D 0.50 nM E 0.73 nM 3) Virus capsid stability Virus have a capsid that is made of many copies of one or a few proteins. These assemble in a regular pattern, where they are held together by non-covalent bonds. In the host cell, where the virus is manufactured, the cytosolic concentration of the capsid protein(s) is high, and the law of mass action favors virus assembly. However, once the virus is released from the cell the concentration of capsid protein(s) drops to essentially zero, and the capsid should disintegrate. The very existence of viral diseases proves that this is not the case. Why? A The process is controlled thermodynamically, by a negative free energy ∆G. B Disassembly would lower the entropy ∆S. C The process is controlled kinetically by a high activation energy ∆E a D The process is controlled by a high heat of reaction ∆H. E The law of mass action does not apply to living systems. 4) Elimination of drugs from the body When a drug enters the body, it can distribute only through part of it. For example, hydrophilic drugs will dissolve in cytosol and interstitial fluid, but not in bones or body fat. The part of the volume of the body available to a drug is called its distribution volume, measured in L. Elimination of a drug from the body is often, but not always, a first order process. A drug (molecular mass 1000 Da) has a distribution volume of 50 L, and is eliminated with first-order kinetics and a half life period of 2 h. 6 h after injection a blood sample is taken and found to contain 12.5 nM. How much was injected?

23

2.5

Biochemistry and Genetics

A) 1 mg

B) 2 mg

C) 5 mg

D) 10 mg

E) 20 mg

5) Effect of pKa on membrane diffusion The depicted compounds are used as local anesthetics for small surgical procedures (dentistry, stitching of wounds). They act in the protonated form (BH+ ) by binding to and blocking the sodium channel from the cytosolic side of the membrane of nerve cells. From the blood stream they enter the cells by passive diffusion, that is, in their unprotonated (B) form. Which of these compounds acts fastest? H N

N O

Lidocain, pKa = 7.7

O

H N

O

N O

Bupivacaine, pKa = 8.1

N

H2N

Procaine, pKa = 8.9

A Lidocaine B Bupivacaine C Procaine D no difference 6) Follow-up: A.B. Drofnats, MD treats a patient with an abscess. Before draining the abscess he applies topical lidocain creme to prevent pain. Upon starting the procedure the good doctor gets a nose-job from his patient. Why? 7) pH-dependent drug trapping Aspirin® (acetylsalicylate) is an over-the-counter pain killer, which is also used to prevent stroke as it reduces blood clotting. The drug has a pKa value of 3.4. What will be the approximate ratio of the drugs concentration in blood (pH = 7.4) over that in stomach (pH = 1.4)? Assume that there is a concentration equilibrium, that no transport proteins for aspirin exist and that stomach juice and plasma are separated by a simple biomembrane as the diffusion barrier. A 0.0001 B 0.01 C 1 D 100 E 10000

24

Objectives

2.5

2.5. Objectives Students should be able to • define the terms system, energy, enthalpy, entropy, work, heat, temperature, free energy, activation energy, reaction order • Define the equilibrium and dissociation constant of chemical reactions • Describe in qualitative terms the relationship between free energy change and equilibrium constant. • Describe the difference between thermodynamic and kinetic properties of reactions. • use fundamental thermodynamic and kinetic equations to characterize a reaction. • discuss how le Chatelier’s principle can be used to change the equilibrium of a reaction • State the importance of the transition state in chemical reactions, its relative free energy content, and the effect of enzymes on the transition state. • define the term catalyst and state the consequences of its presence on a chemical reaction. • Recognize the difference between zero-order and first-order reaction, and state the conditions leading to zero of first order kinetics in catalyzed reactions.

25

3. Amino acids and proteins

... everything that living things do can be understood in terms of the jigglings and wigglings of atoms. (R. Feynman: Lectures in Physics)

3.1. Amino acids

3.1.1. General structure of amino acids

Amino acids contain a carboxy group, an amino group, a hydrogen atom and a variable side chain R (“residue”). These four groups are bonded to a central, asymmetric (chiral) carbon called the alpha-carbon. Only L-amino acids are found in proteins. However, D-amino acids are found in the bacterial cell wall and in several antibiotics. In humans, D-Ser is produced by astrocytes to regulate the response of NMDA-receptors and long-term potentiation. The carboxy group has a pKa close to 2 while the amino group pKa ranges from 9 to 10. Thus, amino acids can exist in different protonation states: COOH

H3N

+

CH R pH = 1

COO

H3N

+

CH R

pH = 7

-

COO

H2N CH R pH = 11

At pH = 7, amino acids exist as zwitterions - molecules that possess both a positive and a negative charge.

27

3.1.2

Biochemistry and Genetics

Figure 3.1.: Structure of amino acids. Top left: Amino acids can form zwitterions (Zwitter (Ger.) = hermaphrodite) with a positive and a negative charge. Top right: Because the α-carbon bears 4 different substituents, it is chiral (exception: glycine where R = H). Bottom: Naming convention of carbon atoms in an amino acid. Figure taken from [Buxbaum, 2007]. O

C

R

OH

O

H2N C H

H3N

+

R

C

O

C H CO

R

H

N

NH2 ,

O α H2N C C C C C C H2 H2 H2 H2 H OH ε

δ

γ

β

3.1.2. The 22 amino acids in proteins

Also some of the amino acid side chains are ionizable1 : Group Amino Acid pKa Carboxy Glutamate, aspartate 4.0 Sulfhydryl Cysteine 8.0 Selenol Selenocysteine 5.2 Phenolic OH Tyrosine 10.0 Guanidino Arginine 12.5 Amino Lysine 10.8 Imidazole Histidine 6.0 The number and pKa values of ionizable groups in a molecule can be determined experimentally by a titration curve, which plots solution pH as a function of increasing quantity of added base (or acid). Each titration plateau indicates an ionizable group and its position shows the pKa to that group. Ionizable groups “buffer” the pH of a solution because they release or absorb protons at pH values close to their pKa . You have to know the (approximate) structures of the 22 amino acids (see fig. 3.2). The amino acid side chains can engage in a number of non-covalent interactions and covalent bonds: 1

pKa values depend on the solvent environment, and vary widely in active sites of enzymes and unique protein environments.

28

3.1.2

The 22 amino acids in proteins

Figure 3.2.: Structures of the 22 amino acids encoded by genes. Acidic groups are marked red, basic blue. Post-translational modifications can significantly change the properties of amino acids in proteins. Sec and Pyl are rare amino acids encoded by alternatively used stop-codons (see the genetic code in fig. 34.4 on page 632). Picture taken from [Buxbaum, 2007]. O H 3N

O

C

+

CH

H Glycine (Gly, G) O H 3N

O

O

C

+

H 3N

CH

H3N

+

O

H3N

+

CH3

CH3

O H3N

+

O H3N

CH

+

O

Threonine (Thr, T)

O

C

H 3N

CH HC OH

HC CH3

+

O

CH

H 3N

+

HC CH3

CH2

CH2

HC CH3

CH3

CH3

Leucine (Leu, L)

O

Isoleucine (Ile, I)

Selenocystein (Sec, U) O

O

C

H3N

CH

O

O

C

+

H3N

CH

+

C

O

CH

CH2

CH2

CH2

C

C

C S CH3 H2

O

Asparagine, (Asn, N)

O

O

C

O

H2N

Aspartic acid (Asp, D)

O

C

CH C Se H2

O

C

O

C

+

C SH H2

Cysteine (Cys, C)

O

Valine (Val, V)

H3N

CH

Serine (Ser, S)

O

CH

O

O

C

+

C OH H2

Alanine (Ala, A)

C

H3N

CH

C H H2

O

O

O

C

+

H 3N

CH

+

O

C

O

CH

CH2

CH2

CH2

CH2

C

Methionine (Met, M)

H 3N

+

Glutamic acid (Glu, E)

CH CH2 CH2

NH2

O

O

CH2

C

O

C

NH

Glutamine (Gln, Q)

C H2N

+

N H2

Arginine (Arg, R) O H 3N

+

O

O

C

H3N

CH

+

CH2 CH2

CH2

CH2

CH2

CH2

+

NH C

N

+

CH

CH2

N H3

H3N

O

CH2

Lysine (Lys, K)

O

C

O

O

C CH

H 3N

+

O

O

C

H 3N

CH

+

CH3

O

Pyrrolysine (Pyl, O)

CH CH2

CH2

CH2

C

O

NH

Phenylalanine (Phe, F) O H2N

+

C C

O Tyrosine (Tyr, Y)

O

O

H H3N

+

Tryptophan (Trp, W)

O

C CH

CH2

NH Proline (Pro, P)

H

N

+

Histidine (His, H)

29

3.1.3 Amino Acid

Biochemistry and Genetics

Hydrophobic Hydrogen interaction bond Glycine (Gly, G) (+) Alanine (Ala, A) + Valine (Val, V) ++ Leucine (Leu, L) +++ Isoleucine (Ile, I) +++ Serine (Ser, S) + + Threonine (Thr, T) ++ + Cysteine (Cys, C) + (+) Selenocysteine (Sec, U) + ++ Methionine (Met, M) ++ Phenylalanine (Phe, F) +++ Tyrosine (Tyr, T) ++ + Tryptophan (Trp, W) ++ + Aspartate (Asp, D) ++ Asparagine (Asn, N) +++ Glutamate (Glu, E) + ++ Glutamine (Gln, Q) + ++ Lysine (Lys, K) ++ +++ Pyrrolysine (Pyl, O) +++ + Arginine (Arg, R) + +++ Histidine (His, H) +++ Proline (Pro, P) ++ Ph. = phosphate ester; CHO = glycosidic bond.

Salt bond + + +++ +++ +++ + -

Covalent bond Ph., CHO Ph., CHO Disulfide Ph. CHO -

3.1.3. The pI-value

At some pH an amino acid has the same number of positive and negative charges. This pH is called the isoelectric point (pI) of that amino acid. While pKa is a property of an individual ionizable group, pI is a property of an entire molecule. The pI is calculated from the pKa -values on both sides of the form of the amino acid which bears no net charge.

30

3-letter Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Sec Ser Thr Trp Tyr Val

Amino acid

Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Selenocysteine Serine Threonine Tryptophan Tyrosine Valine

A R N D C E Q G H I L K M F P U S T W Y V

1-letter

MW (Da) 89 174 132 133 121 147 146 75 155 131 131 146 149 165 115 168 105 119 204 181 117

pK1 (−COOH) 2.34 2.17 2.02 1.88 1.96 2.19 2.17 2.34 1.82 2.36 2.36 2.18 2.28 1.83 1.99 2.16 2.21 2.11 2.38 2.20 2.32

pK2 (NH+ 3 ) 9.69 9.04 8.08 9.60 8.18 9.67 9.13 9.60 9.17 9.68 9.60 8.95 9.21 9.13 10.96 9.40 9.15 9.62 9.39 9.11 9.62

pK3 (R) 12.48 3.65 10.28 4.25 6.00 10.53 5.20 13.60 13.60 10.07 6.01 10.76 5.41 2.77 5.07 3.22 5.65 5.97 7.59 6.02 5.98 9.74 5.74 5.48 6.48 3.68 5.68 5.87 5.89 5.66 5.97

pI

Helix propensity 0.00 0.21 0.65 0.43 0.68 0.39 0.16 1.00 0.56 0.41 0.21 0.26 0.24 0.54 3.16 0.50 0.66 0.53 0.49 0.61

Hydropathy (kJ/mol) +1.8 -4.5 -3.5 -3.5 +2.5 -3.5 -3.5 -0.4 -3.2 +4.5 +3.8 -3.9 +1.9 +2.8 -1.6 -0.8 -0.7 -0.9 -1.3 +4.2

115 140 255 230 155

Surface 2 (Å ) 115 225 160 150 135 180 190 75 195 175 170 200 185 210 145

73 93 163 141 105

Volume 3 (Å ) 67 167 148 67 86 114 109 48 118 124 124 135 124 135 90

Abund. (%) 9.0 4.7 4.4 5.5 2.8 6.2 3.9 7.7 2.1 4.6 7.5 7.0 1.7 3.5 4.6 rare 7.1 6.0 1.1 3.5 6.9

Table 3.1.: Properties of the amino acids found in proteins vary widely. This allows amino acids to fulfill different roles in a protein.

The pI-value

3.1.3

31

3.1.3

Biochemistry and Genetics pI = 5.97 _

_ HO O + C H3N CH2

O O + C H3N CH2

pK1 = 2.34

O

pK2 = 9.60

C

O

H2N CH2

pI = 5.97

glycine 12

_

_ HO O +10 C H3N CH2

O O + C H3N CH2

pK1 = 2.34

O

pK2 = 9.60

C

O

H2N CH2

8 glycine

pH

12 6 10 4 8

pH

2 6 0 0

0.5

1 OH-Equivalents (mol/mol)

4

1.5

2

If the amino acid contains only the Carboxy-group C0 and the amino group on Cα , the pI = 3.22 isoelectric point is simply the average of their pKa s: 2

O HO C + H3N CH

_

0 0

pK1 = 2.19

CH2 CH2 HO C O H+O C O H3N CH

pK1 = 2.19

CH2

1.5 2 × (pK1 + pK+O2 ) C=O1/2 × (2.34 H3N CH pKR = 4.25 pK2 = 9.67 CH2 2 pI = CH 3.22 CH2 CH2 _ _ O C O O C O H+O C O _+O C O H3N CH H3N CH pK = 4.25 pK = 9.67

HO

10

O

1 OH-Equivalents (mol/mol)

R

12

CH2 C

_

_

O 0.5 O pI += C1/2 H3N CH

HO

Glutamate

O

O + 9.60) C = 5.97 CH2 CH2

_

O C O _O C O H2N CH

2

CH2

CH2

CH2

CH2

CH2

C

C

C

O

_O

(3.1)

H2N CH

CH2

_O

O

O

8 Glutamate

pH

12 6 10 4 8

pH

2 6 0 0

0.5

1

4

2

1.5 OH-Equivalents (mol/mol)

2

2.5

3

pI = 7.59 _ O O O O 0.5 1.5 2 2.5 3 C C 1 + + OH-Equivalents (mol/mol) pK = 6.00 pK2 = 9.17 R H3N CH H3N CH _

HO O C + H3N CH

0 0

pK1 = 1.82

_ O

C

O

H2N CH

If, however, the amino acidCHhas a negatively CH charged R-group CH (like Glu), we have to identify CH2 2 2 2 H H H H pI =C7.59 C N C N C N N the form with no net charge. This is the second from the left, which has one positive and _ _ _ CH CH CH CH O C O HO C O O C O O C O N N N N one negative charge. TheCHpK side are 2.19HC and 4.25, and the pI is the C C H + aH-values on +either + H + H + pK2 = 9.17 H N CH pK1 = 1.82 H N CH pKR = 6.00 H3N CH3.22. H3N CH average, 2 3 Histidine

CH2 C N

C N

CH 10

C N H + H

CH2

H

C N

CH

C N H + H

C N H

CH2

H

C N

CH

C N H

8 Histidine

pH

12 6 10 4 8 2

pH

32

CH2

12

H

6 0 0 4

0.5

1

1.5 OH-Equivalents (mol/mol)

2

2.5

3

H CH

4

2

0

0.5

1

1.5 OH-Equivalents (mol/mol)

_ HO O C + H3N CH CH2 C N

pK1 = 1.82

O O C + H3N CH pKR = 6.00 CH2

H

C N

CH

C N H + H

3.1.4

The one-letter code3 2 2.5

0

pI = 7.59 _ O O C + pK2 = 9.17 H3N CH CH2

H

C N

CH

C N H + H

C N H

_ O

C

O

H2N CH CH2

H

C N

CH

C N H

H CH

Histidine 12

10

pH

8

6

4

2

0 0

0.5

1

1.5 OH-Equivalents (mol/mol)

2

2.5

3

For amino acids with a positively charged R-group the rational is similar: The third form from the left has one positive and one negative charge. The pKa -values on either side are 6.00 and 9.17, and the pI is the average, 7.59.

3.1.4. The one-letter code Amino acids are abbreviated with the first three letters of their name. However, this takes three bytes in computer data bases. The 26 letters of the roman alphabet are sufficient to code for 22 amino acids, which is a more economical use of computer memory. Since, however, several amino acids start with the same letter (e.g. Ala, Arg, Asp, Asn), we can not simply use the first letter. The following list should help you to remember single letter codes: • Amino acids with a unique first letter: Cys, His, Ile, Met, Ser, Val • Where several amino acids start with the same letter, common amino acids are given preference: Ala, Gly, Leu, Pro, Thr • Letters other than the 1st letter are used for Asn (asparagiN), Arg (aRginine), Tyr (tYrosine) • Similar sounding names: Asp (asparDic acid), Glu (glutEmate), Gln (Qtamine), Phe (Fenylalanine) • The remaining amino acids have letters that do not occur in their name: Lys (K close to L), Trp (W reminds of double ring), Sec (U), Pyl (O)

33

3.2.1

Biochemistry and Genetics

• X for any aa

3.2. Proteins 3.2.1. The peptide bond A peptide bond is formed by the condensation of two amino acids under elimination of water: Carboxy-terminal end

O O

C

C

R´

H3N

+

+ C

C O R´

C

C

O O

O C C R"

R"

C

HN NH

C O

O C

R´

C

C R

O O

O

H2O

O

+

H3N

C

R +

H3N

Aminoacids

H3N

+

Amino-terminal end

Dipeptide

Polypeptide (> 20)

NH H2O

O C C R H3N

+

Tripeptide

Oligopeptide (< 20)

Protein: Polypeptide with biol. function

The product is a dipeptide. Addition of further amino acids to the chain leads to tripeptides, tetrapeptides and so on. Chains of up to 20 amino acids are called oligopeptides (oligo = few), and longer ones polypeptides (poly = many). Proteins are polypeptides with a biological function. Polypeptides range in size from a few amino acids to hundreds or even thousands. Proteins consist either of a single polypeptide chain, or they are formed from separate polypeptide chains called subunits. Some proteins contain other components, so-called prosthetic groups. Peptide bonds are formed between the carboxy and α-amino groups of two amino acids. Each peptide has an amino terminus, conventionally written on the left, and a carboxy terminus, written on the right side. The peptide bond is rigid. Because of mesomery with the C0 =O-bond it has “partial double bond character” (see fig. 3.4). Like the C=C bond, it is planar and cannot rotate. The H and

34

3.2.1

The peptide bond

Figure 3.3.: Aspartam is a peptide used as an artificial sweetener in the food industry. O O H3N

+

C

O

CH2

O

CH3

N CH H CH2

CH COO

C

-

aspartyl-phenylalanine-1-methyl ester (Aspartam®)

Figure 3.4.: Left: The definition of the dihedral angle of the bond between the atoms B and C. The dihedral angle is then the angle between the bonds A-B and C-D. Right: The geometry of the peptide bond. For details see text. Picture taken from [Buxbaum, 2007]. α

+α ω ψ τ

γ

α

φ α

35

3.2.2

Biochemistry and Genetics

Figure 3.5.: Left: Ramachandran plot of the frequency of φ, ψ-values in proteins. Certain pairs of angles do not occur since they lead to clashes between the atoms of neighboring amino acids. Right: At a certain φ, ψ C’=O of the preceding amino acid clashes with the N-H of the following one, we define this pair of dihedral angles as 0°,0°. Figure taken from [Buxbaum, 2007]. all amino acids except Gly and Pro

2000 1800 1600 1400 1200 1000 800 600 400 200 0

2000

1500 freq 1000

500

0

-180

-135

-90

-45 φ

0

45

90

135

-135 180-180

-90

-45

0

45

90

135

180

ϕ

O of the peptide bond are in the trans-configuration. Formally, we express the same idea by saying that the dihedral angle of the peptide bond ω is fixed to 180°. The other two bonds in the polypeptide backbone can rotate. Therefore the polypeptide can fold, bend and twist itself into a variety of shapes. In particular, the dihedral angles of the C0 −Cα -bond ψ and of the Cα −N-bond φ are flexible. However, not all angles are allowed, because some lead to clashes between the atoms of neighboring amino acids. If we look at the frequency of amino acids with given φ, ψ-values (Ramachandran-plot, see fig. 3.5) we see that certain values are not represented at all (forbidden values). The angles φ, ψ which results in a clash between C0n =O and Nn+1 −H is defined as 0°, 0°.

3.2.2. Protein structure Primary structure The sequence of amino acids in the polypeptide chain of a protein is called its primary structure. By convention, the sequence is read from the N- to the C-terminus, i.e. in the direction in which proteins are synthesized in the cell.

36

Protein structure

3.2.2

Figure 3.6.: Subtilisin (PDB-code 1gcl, left) and Chymotrypsin (PDB-code 1oxg, right) are both Ser-proteases, that use the classical catalytic triade (Ser, His, Asp, shown as wire diagram) in the catalytic center to cleave proteins. These amino acids are far apart in the sequence, but close to each other in the folded protein. Both proteins, however, have completely different sequence and secondary structure. This is an example of convergent evolution (“re-inventing the wheel”). The proteins are called iso-enzymes (iso = the same).

Because each position in the primary structure can be occupied by any of the 20 common amino acids, the possible number of combinations is huge. For example, a protein with 100 amino acids has 20100 = 1.3 × 10130 possible sequences. Given that our universe is about 13.7 × 109 a ≈ 4.32 × 1017 s old, creationists have argued that proteins can not have been created by a process of random mutation and selection. This argument is fallacious, however, since it makes the (unspoken!) assumption that the function of a protein can only be met by one particular amino acid sequence. The existence of isoenzymes, proteins with different primary structure but the same function, proves this assumption wrong.

Secondary structure

The secondary structure of a protein is any regular, repetitive folding pattern in the molecule. Only a few secondary structures are energetically possible:

37

3.2.2

Biochemistry and Genetics

Figure 3.7.: Different ways to represent the structure of a protein. For proteins with more than just a few amino acids the space-filling (a) or even the wire diagram (b) become unreadable. If only the backbone of the protein is drawn (c), this can be avoided. However, disulphide bonds then dangle in free space (d). To increase readability, the disulphide bonds are drawn to the backbone instead (e, which of course is chemically wrong!). For larger proteins even backbone diagrams are too crowded, and structural elements are presented in schematic form instead (f). Figure taken from [Buxbaum, 2007].

38

(a) spacefilling

(b) wire diagram

(c) wire + backbone

(d) dangling disulphide bond

(e) disulphide connected

(f) schematic (PDB 1m40)

3.2.2

Protein structure

Figure 3.8.: Signal-peptide for import into mitochondria. Most mitochondrial proteins are encoded in the nucleus, they are synthesized in the cytosol and them imported via a transport system that spans both mitochondrial membranes. An amphipatic α-helix serves as the recognition signal for binding of the nascent protein to the transporter. Note that the helical wheel projection is viewed from the N-terminus. Figures taken from [Buxbaum, 2007].

(a) side-view

(b) helical wheel projection

The α-helix In the α-helix the polypeptide is wound in a counterclockwise spiral around an imaginary axis. Such a spiral is called righthanded, because if you hold your right hand with the thumb pointing from N- towards C-terminus the fingers curl counter-clockwise. There are 3.6 amino acids per turn, each turn is 5.4 Å long with a pitch of 1.5 Å per residue. φ, ψ = −57°, −47°, and the R-groups stick outward. This compact, rod-like structure is maintained by hydrogen bonds between the components of the peptide bonds: Each peptide bond C=O forms a hydrogen bond with the peptide bond N−H four amino acid residues ahead of it. Because all N-termini point in the same direction, an α-helix has a dipole moment and can bind to charged molecules. Proline and glycine don’t fit well into the α-helix. The α-helix is the most common secondary structure in proteins. It occurs both in many fibrous (long, stretched-out) proteins (such as myosin and keratin), and in many globular (compact-shaped) proteins. Often α-helices have a polar side (facing the outside of a protein) and an non-polar one which is buried in the interior (amphipatic helix). Function of α-helices: • An α-helix of 22 amino acids is long enough to span a double membrane. The part of the helix that is inside the membrane consists of hydrophobic amino acids that

39

3.2.2

Biochemistry and Genetics

Figure 3.9.: Keratin is made from coiled-coils of α-helices. Figure taken from [Nelson et al., 2008].

Figure 3.10.: In heptad-repeats (Leu-zipper) (here tropomyosin, PDB-code 1lc2) every 7th aa is Leu → hydrophobic interactions. This leads to specific associations of αhelices by hydrophobic interactions. This is a stereo-diagram, if you look at the images cross-eyed, you will see 3 figures, the middle of which is 3-dimensional (this takes some practice).

40

Protein structure

3.2.2

can interact with the lipid tails of the membrane. Hydrophilic amino acids on both ends interact with the cytosol and the interstitial fluid, respectively. On the cytosolic end you find more positively charged amino acids, but on the extracellular end more negatively charged ones, because the potential of a cell is negative inside (−70 mV). At the interface between the membrane and the aqueous environment one finds predominantly aromatic amino acids and Lys. • Amphipatic α-helices at the N-terminus of a protein serve as recognition sites for the import into mitochondria. Every 4th or 5th amino acid is positively charged, so that all positive charges are in the same quadrant of the helix (see fig. 3.8). • Two α-helices wound around each other form a coiled coil. Keratin consists of such coiled-coils (see fig. 3.9). These are held together by disulphide bonds. Breaking these with thioglycolic acid is the basis of the permanent wave. • Heptad-repeats (Leu-zippers) are α-helices where every 7th amino acid is leucine. Such helices associate because of hydrophobic interactions between the Leu-residues, allowing for specific dimerization of proteins. Some DNA-binding proteins have this structure. The β-sheet In the β-pleated sheet, the polypeptide backbone is stretched out. Different portions of the protein are aligned in a parallel or antiparallel fashion, forming hydrogen bonds between a N−H group of one strand and a C=O-group in a neighboring strand. This gives rise to large, blanket-like structure. The main difference between α-helix and β-pleated sheet is that in the α-helix hydrogen bonds occur between residues of the same helix, while in a β-pleated sheet they occur between residues of neighboring strands. Nevertheless, a single β-strand is stable since the amino acids in this extended structure have plenty of “wiggling” space without running into steric hindrance (look up the coordinates in the Ramachandran plot!), resulting in entropic stabilization. The R-groups poind up- and downwards in turn, making amphipatic sheets with polar and non-polar or positive and negative faces possible. The entire sheet is rarely flat, but has a right-handed twist, in extreme cases forming a β-barrel. In an anti-parallel β-sheet the stands change direction, going in turn from N → C and vice versa. They are usually joined together by β-turns (see later). φ, ψ = −138°, 137°. In a parallel β-sheet the N-termini of all strands point in the same direction, φ, ψ = −116°, 111°. The hydrogen bonds are oblique to the strand direction, hence the parallel βsheet is less stable than the anti-parallel. The strands in a parallel β-sheet are often joined by α-helices, which form the “return-leg”. Silk-protein is an important example for the use of β-sheets in biologically important structures. Since the amino-acids within a β-strand are already in an extend conformation, silk shows little elasticity and has an extremely high tensile strength, as any extension

41

3.2.2

Biochemistry and Genetics

Figure 3.11.: Anti-parallel β-pleated sheet (here PDB-code 1qjp). Neighboring strands have different directions, and are joined by β-turns. Hydrogen bonds holding the sheet together run vertically to the strands.

Figure 3.12.: Parallel β-pleated sheet (here PDB-code 2v9s). All strands have the same direction, the “return-legs” are either α-helices or coils. Hydrogen bonds holding the sheet together run obliquely to the strands.

42

3.2.2

Protein structure

would require breaking covalent bonds. On the other hand, the strands are held together by hydrogen bonds only, giving silk cloth this wonderful soft flow. The PII (syn.: poly-Pro or polypeptide II) helix is a left-handed helix with three residues per turn and φ, ψ = −70°, 140°. Like the single β-strand, it is stabilized by entropy, not by hydrogen bonds. Pro frequently occurs in this structure, but not all PII helices contain Pro.

Table 3.2.: Collagen-related inherited diseases. FACIT = fibrillar associated collagens with interrupted triple helices. OMIM (Online Mendelian inheritance in man) is a catalogue of inherited diseases at http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim, which you should bookmark. Type

composition

function

I

α1(I)2 α2(I)

skin, bones

II

α1(II)3

III

α1(III)3

IV

α1(IV )...α6(IV )

tendon,

cartilage, vitreous body skin, muscle, vessels, fetus basal lamina

source of tumstatin

V

α1(V ), α2(V ), α3(V )

interstitium, placenta

OMIM

Gene

Location

diseases

+120150

COL1A1

17q21.3-q22

*120160

COL1A2

7q21.3-q22.1

+120140

COL2A1

12q12-q13.2

OI, EDS, osteoporosis OI, EDS, Marfan, osteoporosis chondrodysplasia

*120180

COL3A1

2q31-q32.3

EDS

*120130

COL4A1

13q34

porencephaly, brain small vessel disease

*120090 *120070

COL4A2 COL4A3

13q34 2q36-q37

*120131

COL4A4

2q35-q37

*303630

COL4A5

X

*303631

COL4A6

Xq22

120215

COL5A1

9q34.2-q34.3

*120190 *120216

COL5A2 COL5A3

2q14-q32 19p13.2

Alport syndrome, benign familial hematuria Alport syndrome, benign familial hematuria Alport syndrome diffuse leiomyomatosis EDS EDS

43

3.2.2

Biochemistry and Genetics

Type

composition

function

OMIM

Gene

Location

diseases

VI

α1(V I), α2(V I), α3(V I)

interstitium, intima

*120220

COL6A1

21q22.3

*120240

COL6A2

21q22.3

*120250

COL6A3

2q37

Bethlem myopathy, Ullrich congenital muscular dystrophy Bethlem myopathy, Ullrich congenital muscular dystrophy Bethlem myopathy, Ullrich congenital muscular dystrophy Epidermolysis bullosa, toenail dystrophy

VII

α1(V II)3

below basal lamina of skin

*120120

COL7A1

3

VIII

α1(V III), α2(V III)

hemidesmosomes in skin

*120251

COL8A1

3q11.1-q13.2

*120252

COL8A2

1p34.2-p32.3

120210

COL9A1

6q12-q14

*120260

COL9A2

1p33-p32.2

*120270

COL9A3

20q13.3

120110

COL10A1

6q21-q22

120280

COL11A1

1p21

120290 *120320 *120350

COL11A2 COL12A1 COL13A1

6p21.3 6q12-q13 10q22

*120324 *120325

COL14A1 COL15A1

8q23 9q21-q22

*120326 +113811

COL16A1 COL17A1

1p35-p34 10q24.3

*120328

COL18A1

21q22.3

*120165

COL19A1 COL20A1

6q12-q13 20q13.33

IX

α1(IX), α2(IX), α3(IX)

X

α1(X)3

XI

α1(XI), α2(XI)

XII XIII

α1(XII)3 α1(XIII)3

XIV XV

α1(XIV )3 α1(XV )3

XVI XVII

α1(XV I)3 α1(XV II)3

XVIII

α1(XV III)3

XIX XX

α1(XIX)3 α1(XX)3

44

cartilage, reous (FACIT)

vitbody

hypertrophic + mineralizing cartilage cartilage

FACIT transmembrane protein FACIT proteoglycans in basal lamina FACIT hemidesmosomes in skin source of endostatin FACIT

Fuchs endothelial corneal dystrophy Stickler Syndrome, Multiple epiphyseal dysplasia Multiple epiphyseal dysplasia Multiple epiphyseal dysplasia, Intervertebral disc disease

Collagenopathy type I+II

Epidermolysis bullosa Knobloch syndrome

3.2.2

Protein structure

Figure 3.13.: Collagen (PDB-code 1cag). To make the tight association between the three strands clearer, one each is drawn space-filling, as wire diagram and as carbonbackbone. Note the repeating Gly-X-Pro (yellow, green, brown; with X often hydroxy-Pro) sequence. Marked in blue is a Gly→Ala mutation that prevents a close fit and destabilize the molecule. Such mutations cause for example Ehlers-Danlos-syndrome.

Type

composition

function

OMIM

Gene

Location

XXI XXII

α1(XXI)3 α1(XXII)3

FACIT cell adhesion in the lung?

*610002 *610026

COL21A1 COL22A1

6p12.3-p11.2 8q24.3

XXIII XXIV

α1(XXIII)3 α1(XXIV )3

610043 610025

COL23A1 COL24A1

5q35.3 1p22.3-p22.2

XXV

α1(XXV )3

*610004

COL25A1

4q25

XXVI XXVII XXVIII

α1(XXV I)3 α1(XXV II)3 α1(XXV III)3

*608927 *608461 609996

EMID2 COL27A1 COL28A1

7q22.1 9q33.1 7p21.3

embryonic bone formation cell membranes in brain embryonal tissues all tissues neural and connective tissue

diseases

Alzheimer

The most important example for the PII -helix are the collagens, which consist of 3 PII helices wound around each other (hetero- or homo-oligomer). In the human genome there are 42 collagen genes, which encode for 28 known collagen types. Of these types I, II and III are the most important. Each of the three molecules in collagen has 1050 amino acids, with the sequence Gly-X-Pro. The sharp angle of the Pro peptide bond allows the sharp turn in the molecule, and the small R-residue of Gly (only a H) allows the 3 protein molecules to wrap around each other. If only a single of the Gly-residues in one of the collagen chains is mutated, wrapping is no longer possible, leading to osteogenesis imperfecta (brittle bone disease, collagen I), to Ehlers-Danlos-syndrome (collagen I, III or V) with too brittle or too elastic ligaments and death by vascular or organ rupture, epidermolysis

45

3.2.2

Biochemistry and Genetics

Figure 3.14.: β-turn (here in PDB-code 1qiv) are most common between the strands of an anti-parallel β-sheet.

bullosa (blistering of skin, collagen XVII), or to Alport-syndrome (collagen IV, kidney and hearing defects). Heating turns collagen into gelatine. The dissociation temperature of collagen is influenced by the hydroxylation of proline. Vitamin C (ascorbic acid) is required for the correct function of Pro-hydroxylase. In scurvy, the dissociation temperature of collagen drops below the body temperature of 37 °C, explaining the connective tissue weakness typical for this condition. Apart from collagen, PII -helices also occur in SH3-domains, which occur in proteins involved in signal transduction (more about this in the course on hormone effects). The hairpin turn Hairpin turns allow the protein to fold back onto itself in a 180° angle. This is required, for example, in anti-parallel β-sheets. Since the C=O- and N−H-groups of a turn are not all involved in hydrogen bond formation within the protein, they are often surface-exposed and interact with water. They may also occur in the catalytic center of enzymes, where they are involved in substrate binding. Because of its small size, Gly is often found in turns. There are two types of hairpin turn; the β-turn with 4 amino acids (frequent, φ, ψ = −56°, 137°.), and the rare γ-turn with 3 amino acids. The coil A coil is basically any secondary structure except those mentioned above. It is important to realize that all the amino acids in a coil have defined positions, making the term “random coil”, often found in textbooks, false. Coils give the protein flexibility, which allows for conformational changes. Since their peptide bonds are not involved in intraprotein hydrogen bonding, they are often exposed to interact with water, small ligands or other proteins. Coils tend to tolerate mutations better than other structures and are

46

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therefore hot-spots for evolution. In the chapter on immunoproteins you will learn that it is coils that give antibodies their specific binding properties. Tertiary structure describes how the elements of secondary structure of a protein are organized in 3D-space. Tertiary structures are formed by hydrophobic interactions of amino acid side chains. Typical globular proteins have a core of hydrophobic side chains, while hydrophilic side chains are on the surface where they interact with water or with other proteins. If hydrophobic residues were exposed to water, the water would have to form an ordered cage around them, which would decrease the entropy of the system. van der Waals-interactions, which are fluctuating dipole interactions with a bond energy of 4–17 kJ/mol. The bond length is ≤ 4 Å. hydrogen bonds, which are interaction between permanent partial charges. The bond length is about 3 Å, the bond energy is 2–6 kJ/mol if both partners are partially charged and up to 21 kJ/mol if one partner is fully charged. If the distance between the partners is too large, an indirect hydrogen bond may be formed where water acts as a bridge. salt bridges, which are interactions between fully charged groups. The bond length is 2.8 Å and the bond energy normally 10–30 kJ/mol, but can be significantly higher if both groups are buried in a hydrophobic environment. Disulfide bonds, which are formed by an oxidative reaction between two Cys residues after folding of the protein into its higher-order structure. They may occur between two Cys residues in the same polypeptide (intra-chain), or between different polypeptides (inter-chain). Disulfide bonds are uncommon in cytosolic proteins, but may be formed in the oxidizing environment of the endoplasmic reticulum (ER). They are therefore present in secreted and plasma membrane proteins. Bond length is 2.2Å and bond energy 167 kJ/mol. Coordination around cofactors Several amino acids in a protein are involved in the coordination of metal ions (Ca, Zn, Fe, Mg, Na, K) or other cofactors, such as heme or FAD. Tertiary structure and evolution Domains are independently folding parts of a protein, which can often be isolated by gentle proteolysis under retention of function. Evolutionary they are often parts of eukaryotic proteins which originated from the fusion of a prokaryotic operon into a single protein. Most domains are between 50 and 250 amino acids long, shorter domains would be devoid of function and longer once may be unable to fold properly.

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Figure 3.15.: If you have done the logical thing and plotted the φ, ψ-values for the various secondary structures into the Ramachandran-plot you should have gotten something like this. At the top of the diagram are the extended structures (parallel and antiparallel β-sheets, PII -helix and turn). The big peak below is at the coordinates of the α-helix, the small peak to the right is the left-handed αl -helix, a rare structure that proteins can adopt only for a few amino acids.

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Motives are the folding patterns of proteins. Evolutionary they are often, but not necessarily, the result of common ancestry. Folding patterns are much more stable during evolution than amino acid sequences; we can recognize common ancestry in cases where there is no longer a statistically recognizable sequence homology. This is understandable: a change of an amino acid may not result in much functional change, but a change in protein folding certainly would. Because the folding patterns are so illuminating, efforts have been made to categorize proteins by tertiary structure. The most important one is structural classification of proteins (SCOP). Lesser known attempts like class architecture topology homology (CATH) and families of structurally similar proteins (FSSP) give largely similar results. SCOP recognizes the following classifications: Class relative content of α-helix and β-strand. Fold Major structural similarity, identical structural elements and topological connection. Superfamily Domains with common folding pattern and similar function. Probable common evolutionary origin despite low sequence similarity. Family Proteins have high structural and sequence similarity. They clearly have a common ancestor. For our purposes, only the class is important: All α proteins contain only α-helices, or their content of β-strands is insignificant. Example: hemoglobin (1hga) All β proteins contain only β-strands, or their content of α-helices is insignificant. Example: Immunoglobulins (ifc2) α/β Proteins with alternating α-helices and β-strands → parallel β-sheets. Example: Thioredoxin (2trx) α + β Proteins with segregated α-helices and β-strands → anti-parallel β-sheets. Example: Lysozyme (132l) Multi-domain proteins have domains belonging to different classes. Example: DnaK (1dkz) Membrane and cell surface proteins (excluding the immune system). Often the transmembrane domains are α-helices (e.g. bacteriorhodopsin, 1m0k), but β-barrels (e.g. OmpA, 1qjp) may also occur. Small proteins usually dominated by metal ligands, heme, disulphide bridges. Example: insulin (1mso) Coiled coil proteins α-helices wound around each other. Example: Fibrinogen (1m1j)

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Biochemistry and Genetics

Figure 3.16.: Intrinsically disordered proteins exist in 3 states (“the trinity”): folded, molten globule and disordered. Each of these may have its own biological function.

ordered (solid-like)

molten globule (fluid-like)

extended (gas-like)

Intrinsically disordered proteins contain relatively long coil segments, possibly interspersed with short segments of other secondary structures. Their tertiary structure is either an extended strand or a molten globule. When these disordered segments interact with other proteins, they bind under re-folding to a different secondary and tertiary structure with the partner protein serving as a folding template. We do not yet fully understand these sequences, it appears that stretches of many identically charged amino acids keep that part on the surface of the protein as an extended coil, preventing the hydrophobic interactions required for folding. A large number of hydrophobic amino acids (more than 5 in a row) result in considerable entropy gains upon binding to a partner, these segments often form molten globules. In enzymes, intrinsic disorder is relatively rare (although examples exist, e.g., chorismate mutase). It is an important feature of many regulatory proteins, however. These regulating factors may bind to many regulated proteins, and their structure is different with each partner. On the other hand, a regulated protein may bind different regulating ones, forcing each of them into similar structures. Thus the intrinsically disordered segments allow one-to-many and many-to-one relationships between regulating and regulated proteins and effectively dissociate specificity and affinity: folding upon binding results in loss of entropy, this reduces ∆G and hence increases Kd . As a result, binding is readily reversible, allowing quick termination of a signalling event. The surface area available for binding is larger 2 (70 Å per residue), at the same time the flexibility to accommodate binding is increased. Binding is often regulated by post-translational modification of proteins like phosphoryla-

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tion (see later), this can change Kd by several orders of magnitude and generates molecular switches. Disordered sequences are very sensitive to proteolytic attack. This creates technical problems for the researcher and is certainly one of the reasons why the significance of these segments are only now more fully appreciated. It also leads to rapid degradation of the protein inside the cell, an important feature for regulatory function. The interaction between partners is robustly encoded in the domains and maintained during evolutionary events which modify, rather than abolish, an interaction. Experiments indicate that early enzymes probably were more disordered and hence flexible, by becoming more structured enzymes specialized on a particular function. Because of their importance in regulation, intrinsically disordered proteins are more frequent in eukaryotes (more than 500 have been identified) than in prokaryotes. An exception to that rule are the Apicomplexa, who have unusually many intrinsically disordered proteins. Important pathogens, like the malaria parasite, belong into that clade. How pathogenicity depends on protein disorder is a topic of current research. Amyloid-formation – a process causing various debilitating diseases – can be understood in similar terms (see later). Quaternary structure Quaternary structure describes how several polypeptide chains (“subunits”) come together to form a single, functional protein. The subunits are held together by the same forces that we have discussed for tertiary structure. Depending on the number of subunits in a protein, we speak of mono-, di-,...,oligo- or polymers. If all subunits are identical, we precede this by homo- and else by hetero-. Sometimes different subunits come together to form a protomer, and several of these then form the functional protein. An example would be hemoglobin, which is a diprotomer, each protomer is a heterodimer composed of an α- and a β-subunit. Determination of protein structures Astounding progress has been made in the determination of protein structures over the last few decades. The protein data base (PDB) started in 1971 with only 7 entries. As of April 2010 almost 65 900 structures have been submitted, and of those, about 3900 were non-redundant. Unfortunately, only 234 membrane protein structures are available, even though membrane proteins make 70 % of the 500 or so drug targets. Difficulties in membrane protein structure determination seriously impedes drug development. Several methods can be used to determine the structure of a protein, each with its own advantages and disadvantages:

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Biochemistry and Genetics

X-ray crystallography Soluble proteins are crystallized, then investigated by X-ray diffraction. No size limit (whole virus). Nuclear magnetic resonance Atomic distances within a molecule are measured in solution. Size limit ≈ 20 kDa. Electron microscopy uses 2D-crystals or molecules sorted by orientation to calculate “average” picture. Resolution limited to 10–20 Å, but works on insoluble proteins. All these methods require large amounts (≈ 100 mg) of pure protein. Since primary structure determines folding (Anfinsen-hypothesis), folding of a protein with a known amino acid sequence should be predictable. People have tried to identify which combinations of amino acids tend to occur in which secondary structure, and then predict the structure of an unknown protein. In practice, success is still very limited because protein structure is not only determined by short-range interactions between neighboring amino acids, but also by interactions between amino acids which are far apart in the primary structure, but come close together in the final tertiary structure. If the structure of a similar protein is available, “threading” is more promising. In theory, one should be able to calculate the structure by taking all possible bond energies into account and minimizing the free energy ∆G of the protein in a computer. However, these “ab initio” (Lat.: from first principles) calculations require way more computational power than even modern supercomputers have (for computing experts: the problem is NP-complete). Protein folding and denaturation Protein chemists work from the assumption that all the information coding for the native 3D-structure of a protein is contained in its amino acid sequence, and that no extraneous information is required to direct folding. This is known as the Anfinsen-hypothesis (N.P. 1972). Levinthal’s paradox: Assume a protein with 100 peptide bonds, each of which can assume 6 stable conformations (α-helix, β↑↑ -sheet, β↑↓ -sheet, PII -helix, turn, coil). Since each of these states is characterized by a φ, ψ-angle pair, this results in 26 = 64 possible angles per peptide bond and 10064 = 10128 for the entire protein (note that this is an underestimate!). Rotation around a σ-bond takes about 10−13 s, thus folding by random testing of all possible angles would take 10128 × 10−13 s = 10128−13 s = 10115 s. Our best estimate for the age of the universe is 13.7 × 109 a ≈ 4.32 × 1017 s. Proteins therefore should never fold. In reality, folding is a rapid process; in E. coli at 37 °C a 100 amino acid protein folds in about 5 s. During folding hydrophobic residues are buried in the interior and hydrophilic

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Protein structure

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Figure 3.17.: Protein folding reduces free energy (G). The native structure is the one with the lowest free energy. However, proteins may get kinetically trapped in local minima of the energy landscape. At the same time the entropy (S) is also reduced, symbolized by the width of the funnel.

residues appear on the outside of the protein, resulting in a compact “molten globule” structure. This brings amino acids so close to each other that the formation of hydrogen bonds between peptide bonds gives rise to secondary structure.

Energetics and kinetics of protein folding Amino acids undergoing folding have a choice of undergoing interactions with either P P other amino acids or with water. Thus only the difference ∆Gfolding = ∆Ga−a − ∆Ga−w is available to stabilize the native structure of proteins. Although folding decreases the enthalpy (H) (which stabilizes native structure), it also decreases the entropy (S), which tends to destabilize native structure. Thus protein folding is a compromise between forces, and the actual stabilization energy is only about 20–40 kJ/mol, about 10× the thermal energy at room temperature (2.5 kJ/mol). This marginal stability of proteins has a good side however: it allows protein flexibility required for ligand binding and enzymatic activity. Although protein folding is a spontaneous process driven by thermodynamic forces and hence, given enough time, all proteins will eventually arrive at their native structure, kinetically the process can be trapped in local minima in the energy landscape. Such metastable intermediates would expose hydrophobic patches on their surface, which leads to protein ag-

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Biochemistry and Genetics

Figure 3.18.: Reaction cycle of adenylate kinase, an enzyme that catalyzes the reaction 2 ADP * ) ATP + AMP. Substrate binding and product release are accompanied by considerable movement of the protein, with coils serving as hinges. Such movement is possible only because the stabilization energy of protein folding is relatively low and can be overcome by the binding energy for the substrates. Film taken from [Vonrhein et al., 1995].

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gregation. Note that proteins in the cytosol are very closely packed at about 300–400 mg/ml. The average distance of protein molecules is just one protein diameter, and the space between them is filled with water, salts and metabolites. As opposed to quaternary structure, such aggregates have no reproducible structure and no biological function. On the contrary, they may interfere with cellular function (see later). Molecular chaperons and chaperonins Cells have two lines of defences against misfolded proteins: Molecular chaperons bind to unfolded proteins and prevent their aggregation until these proteins can achieve folding. Binding/unbinding cycles of chaperons may or may not require the hydrolysis of ATP. Examples: Hsp70, Hsc70, crystallins. Molecular chaperonins use the energy of ATP-hydrolysis to actively unfold misfolded proteins, giving them a second chance to arrive at the proper fold. Example: GroES/GroEL. Unfolding occurs in a “beaker” formed by the chaperonin, in which the client protein can try to refold without disturbance from other proteins (at “infinite dilution”). The beaker has a diameter of about 45 Å, enough to contain proteins (or protein-domains) of up to 60 kDa. Note: Neither chaperons nor chaperonins actively fold proteins, they merely protect them against aggregation during the folding process. Protein denaturation The normal interactions that maintain the higher-order structures of proteins are weak and can be disrupted easily. Heat denaturation occurs when the protein is heated to more than 40–70 °C. This results in loss of biological activity and precipitation. Renaturation is sometimes possible with small proteins (ribonuclease, lysozyme) under laboratory conditions, but denaturation is irreversible in the real world (example: boiled egg). Once a few bonds within the protein are broken by the increasing movement of the protein chain the protein is destabilized and further bonds are broken more and more easily. Hence heat denaturation of proteins is a highly cooperative process. Humans die if their body temperature exceeds 42 °C because key proteins are denatured. Also other insults can denature proteins: Strong acids and bases denature proteins by disrupting ionic interactions. Organic Solvents can denature proteins by disrupting hydrophobic interactions. Proteins are not soluble in organic solvents. More water soluble solvents (e.g. ethanol or acetone) bind water and thus reduce the concentration of water available to the protein. Detergents also disrupt hydrophobic interactions. They can denature proteins without precipitating them.

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Biochemistry and Genetics

Figure 3.19.: The pathway from genome to phenome is studied at different levels with different methods. The resulting complex data can only be handled by advanced computer techniques. Figure from [Buxbaum, in press].

Small hydrophilic substances , such as urea can denature proteins when they are present in very high concentration. Salts precipitate proteins because they reduce the concentration of water available to maintain protein structure. Heavy metal ions (lead, mercury) bind to carboxylate or sulfhydryl groups of proteins. That’s why they are toxic! The covalent bonds in proteins are more robust, but peptide bonds are hydrolyzed by heating in strong acids and bases, and by proteolytic enzymes. Disulfide bonds are cleaved by reducing and oxidizing agents.

Post-translational modification of proteins The human genome contains ≈ 30 000 genes. mRNA-processing (alternative splicing, mRNA editing etc.) results in ≈ 3 mRNAs per gene. Post-translational modification of the proteins produced from them creates ≈ 10 different protein species from each mRNA. Thus the human proteome consists of ≈ 106 proteins, with different function, regulation, destruction... Glycosylation is the process of enzymatic transfer of oligosaccharide (sugar) trees to the proteins. They are affixed either to the OH-groups of Ser or Thr (O-linked) or to the acid amide group of Asn (N-linked). Other amino acids (Arg, Tyr, Trp, Hyl, Hyp) are involved much less frequently, e.g., collagen. Addition occurs in the ER and the Golgi-apparatus to

56

Protein structure

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the extracellular domain of membrane proteins and to secreted proteins. Cytosolic proteins are rarely glycosylated. In bacteria, glycosylation occurs in the periplasm. Glycosylation is required for proper protein folding. Glycosylation inhibitors are used as antiviral drugs (e.g. nojirimycin or desoxynojirimycin). Sugar-trees are also required as “address labels” in the intracellular transport of proteins between compartments. For example, in I-cell disease enzymes of the lysosome can not be transported into this organelle because the enzyme which transfers the sugar mannose-6-phosphate to them is defective. They are excreted into the blood stream instead and, as a consequence, the lysosomes are non-functional. On the cell surface, the sugar-trees of membrane proteins serve as recognition sites for cellcell-interactions, as immunological determinants (blood group antigens, see fig. 3.20) and – since everything has to have a downside too – as docking sites for bacteria and virus. Clostridium ssp. infection Glycosylation of Rho GTPases by bacterial enzyme results in loss of a nucleotide binding site Type II diabetes glutamine:Fru-6-P amidotransferase stimulation leads to increased [GlcN Ac], transfer of GlcNAc to regulatory proteins involved in insulin resistance Congenital disorders of glycosylation failure to produce dolicholpyrophosphate-sugar tree, no N-linked glycosylation → neurological defects, failure of maturation of N-linked glycoproteins Leucocyte adhesion deficiency GDP-Fuc is not produced or not imported into Golgi, no fucosylation of EGF-motives occurs. Paroxysmal nocturnal hemoglobinuria The GPI-anchors of proteins in granulocytes and B lymphocytes are missing.

Glucosylation This is one of those points where you really have to watch your mouth: Although glucosylation and glycosylation are spelled only with one different letter, they denote completely different processes. Glycosylation is an enzymatic process and carefully orchestrated by the cell. Glucosylation is a spontaneous process that does not require any enzymes. The velocity of this reaction depends on the concentration of glucose in the blood. This has a direct medical application: In diabetics, the concentration of glucated hemoglobin (HbA1c ) depends on the average blood glucose concentration during the lifespan of an erythrocyte (about 3 mo). The formation of advanced glycation end-products (AGE) from glucosylated proteins is thought to be involved in aging and in long-term diabetic damage.

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Biochemistry and Genetics

Figure 3.20.: The AB0 system of blood group antigens. Sugar trees are added to both proteins (O-linked) and lipids. All people can make the 0-antigen. Transfer of an additional GlcNAc-residue creates the A-, of an additional Gal-residue the B-antigen. People who have the GlcNAc-transferase only have the blood group A, while those who have the Gal-transferase only have blood group B. People with both enzymes have blood group AB, those with neither have blood group 0. Picture taken from [Buxbaum, 2007].

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Protein structure

Figure 3.21.: Glucosylation of proteins by the aldehyde group of glucose proceeds via an unstable Schiff-base and Amadori-rearrangement to a stable ketosamine. During roasting, this is converted into caramels via the Maillard-reaction. These are responsible for the taste of cooked food. Ketosamine may also be converted to advanced glycation end-products (AGE) by Strecker-degradation. HC

O

+

HC OH

COOH H

OH

COOH

H2O

+

R'

R

H COOH +

HC N CH H HC OH R

H2N CH

HC N CH HC OH R'

R

R Immonium

Schiff base

COOH

CH2OH

COOH

O

H N CH

OH

H

+

R'

OH

HC N CH H C OH R' R Amadori-Rearrangement

OH

Glucosylamine

H2O C

H2C N CH H C O R'

H2C N C H C O R' R Reductone (Caramel)

COOH

O

!

R

Strecker degradation

ketosamine

Maillard-Reaction

Figure 3.22.: The tripeptide glutathione serves as a redox-coupler in our cells. Figure taken from [Buxbaum, 2007]. COOH H2N CH

O

CH2

O

CH2 O

C

N H

C CH

N H

C

OH

CH2

CH2 SH

*OXWDWKLRQH*6+ ( -Glu-Cys-Gly)

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Disulphide bonds Reduction of the SH-groups of two cysteine residues leads to the formation of a covalent bond. The cell uses the tripeptide glutathione (see fig. 3.22) as reducing agent: −SH + HS− + GSSG * ) −S−S− + 2GSH. Disulphide bond formation does not happen in the reducing environment of the cytosol, but in the ER (or the bacterial periplasm) which is oxidizing. Special enzymes, protein disulphide isomerase (PDI), make sure that the right Cys residues undergo disulphide bond formation. When cytosolic proteins are used in the laboratory one has to make sure that their SHgroups are not oxidized by air oxygen, which would lead to inactivation. The buffers therefore usually contain an antioxidant like β-mercaptoethanol or dithiotreitol. Some mucolytic pharmaceuticals, like N-acetylcysteine (ACC), work by breaking S−Sbonds in mucus proteins, decreasing the viscosity of mucus and making it easier to clear from the airways. Bacterially expressed eukaryotic proteins are often miss-folded and precipitate as inclusion bodies because bacteria are less active in disulphide formation than eukaryotes. However, bacteria do have an enzyme operon (Dsb, short for disulphide bond) for formation and isomerization of protein disulphide bonds in their periplasm.

Proteolysis Proteolysis is involved in the activation of proenzymes, for example in the digestive system. Digestive enzymes (e.g. trypsin, chymotrypsin) are produced as inactive precursors, so that the can not harm the cells secreting them. Once released into the intestine, they are activated by cleaving off a part of the enzyme that was blocking the active site. Pro-hormones (e.g. insulin) are activated in a similar manner. On the other hand, proteins no longer needed can be inactivated by proteolysis (e.g. cyclins in cell cycle). Proteolysis may also be used to remove signal peptides. For example, proteins destined for the intermembrane space of mitochondria carry a signal protein for mitochondrial import (see fig. 3.8) which leads to their import into the mitochondrial matrix. There the signal peptide is cleaved off by matrix protease, exposing a second signal directing the proteins export into the intermembrane space through a different transporter. A special form of proteolysis is protein splicing. This reaction is carried out by a protease within the protein itself, the intein. This protease cuts itself out of the protein and rejoins the flanking segments (exteins), and all this without requiring any external proteins, cofactors or sources of energy (like ATP)! Inteins are now used as self-cleaving affinity-tags to make protein pharmaceuticals.

Hydroxylation Protein hydroxylation occurs on Pro and Lys residues, for example in collagen (see section on collagen above for further discussion).

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Another protein regulated by Pro-hydroxylation are the hypoxia induced transcription factors (HIF). These consist of two subunits, α and β. In the presence of oxygen, the α-subunit is hydroxylated on P402 and P564 by HIF-prolylhydroxylases (PDH-1, -2 and -3), leading to their proteasomal destruction. In the absence of oxygen, the α-subunits accumulate and form a complex with β, which binds to hypoxia response elements in the cellular DNA. As a consequence, oxygen consumption of the cell is down-regulated, it can survive low oxygen supply for a longer time. This mechanism may one day be exploited to increase the survival time of organs in infarct or transplantation, e.g., with inhibitors of PDH-1 (currently available inhibitors produces too many side effects due to concomitant inhibition of PHD-2 and -3). Phosphorylation/dephosphorylation The transfer of phosphoryl groups from ATP to the hydroxy groups of Ser, Thr and Tyr (rarely onto His, Asp and Glu) is important for the reversible regulation of enzyme activity. The transfer is catalyzed by protein kinases, the removal by protein phosphatases. Thus the reaction is rapidly reversible at minimal expense for the cell (a single high energy phosphate bond). You will study many examples for this type of regulation in the next couple of months, 1/3 of all proteins in the cell undergo regulatory phosphorylation/dephosphorylation cycles. Acetylation/deacetylation Transfer of acetyl-groups from acetyl-CoA onto the -amino group of Lys by protein acetylases, and their removal by protein deacetylases, are also used for regulation of enzymatic activity. We know much less about acetylation than about phosphorylation. Many DNA-binding proteins are regulated by acetylation, because the acetylated Lys is much less likely to be protonated, hence less likely to bind to the negative charges of DNA. Three classes of deacetylases are known: class I and II hydrolyze the bond with water, while class III deacetylases (sirtuins) use NAD+ , thus their activity depends on the nutritional status of the cell. This is probably the mechanism behind the observation that caloric restriction prolongs the life expectancy of lab animals. Methylation/demethylation Transfer of methyl-groups from S-adenosyl methionine (SAM) onto proteins may also serve regulatory purposes, but we know very little about it. Transfer can be to carboxyl groups, forming methyl esters. This reaction is used to mark damaged proteins for destruction, but also in signal cascades of unknown function. amino groups, forming methyl amines. The function is unknown. There are no N-demethylases, so the modification is permanent. thiol groups, forming thioesters. The function is unknown. Unlike phosphorylation, methylation has never been observed to occur on hydroxyl-groups.

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Figure 3.23.: ADP-ribosylation of proteins. Figure taken from [Buxbaum, 2007]. NH2

O N

H2N Protein

Arg

NH2

+

N

O O

HO

O

P O

N

O O

P O

N

O

O HO

OH

N

OH

NAD+

Cholera toxin

NH2 N Protein

Arg

H N O

HO

O OH

O

O

P

P

O

O

O

N N

O

O

N

O

+

NH2 N H

HO

OH

Addition/removal of hydrophobic tails Addition of palmityl- (fatty acid) groups to internal Cys or Ser myristoyl- (fatty acid) groups to N-terminal Gly farnesyl- or geranylgeranyl (isoprenoids) groups to C-terminal Cys converts cytosolic enzymes to membrane-bound (cytosolic leaflet). Since this is required for the activation of some enzymes, the transferases make possible drug targets (e.g. anti-cancer drugs). ˙ −OH. The Cys must be * R−S−N S-Nitrosylation occurs on Cys-residues: R−SH + NO ) positioned between a basic and an acidic residue (either in primary, tertiary or quaternary structure) because of acid-base catalysis. Nitrosylation serves as an additional pathway for NO regulation besides cGMP-dependent kinases. This pathway too has not been fully explored.

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ADP-ribosylation ADP-ribosylation is used by some bacterial toxins (Vibrio cholerae, Bordetella pertussis) to inactivate cellular proteins. This is the starting point of the pathomechanism of the diseases associated with these bacteria (cholera and whooping cough, respectively).

Deamidation Removal of the acid amide group from Gln or Asn, forming Glu and Asp, respectively. May be followed by racemization (formation of D-amino acids). • catalytic removal by bacterial pathogenicity factors (cytotoxic necrotizing factors) on heterotrimeric G-proteins and small GTPases → GTPase activity is inhibited, the protein can not go from the active GTP-bound to the inactive GDP-bound form. • spontaneous (Asn faster than Gln): age determination in forensic science long lived, low turnover proteins (bone, teeth) archaeology rate constant depends on temperature, humidity, soil pH etc. Hence better suited for determination of relative age within a series of finds.

AMPylation (adenylation) is performed from ATP onto critical Thr hydroxygroups of Rho GTPases and other important regulatory proteins by Vibrio parahaemolyticus Vibrio outer protein S (VopS). This results in depolymerisation of the actin cytoskeleton, affected cells round up. Like in ADP-ribosylation the bacterial toxin uses a readily available energy-rich substrate to disable critical host proteins. Like the ADP-ribosylation from NAD or glucosylation from UDP-glucose AMPylation from ATP is performed by a bacterial A/B toxin to interfere with host defences. The toxins use readily available, energy-rich co-substrates to modify and hence inactivate key proteins. AMPylation however is (at least in bacteria) also used for control of metabolism: The glutamine synthetase of E. coli is controlled in part by AMPylation of Tyr-397, the adenylyl transferase responsible in turn is controlled by uridilylation.

Transfer of peptides Ubiquitin is transferred onto proteins no longer needed, the proteins are then degraded in the proteasome. Ubiquitin is transferred to the -amino-group of lysine, forming an isopeptide bond. There is a whole family of small ubiquitin-like modifiers (SUMO) which are transferred in a similar manner, but about whose function we have little knowledge.

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The relationship between protein structure and function: Green fluorescent protein GFP is produced by the coelenterate Aequorea victoria. It accepts energy from a chemiluminescent protein (which would otherwise produce blue light) and translates it into green light by bioluminescence resonance energy transfer (BRET). The function of bioluminescence in these animals is unknown. Because of its intensive green fluorescence GFP has become a favorite marker in molecular biology. The genetic information for GFP is attached to the gene for the protein under investigation, so that a fusion product is generated. Thus both the amount of target protein produced and its subcellular localization can then be studied by fluorescence video microscopy.

Production of the fluorophore of GFP from 3 neighboring amino acids This process can proceed in the absence of other proteins and cofactors except oxygen. Thus GFP can be used as marker in any aerobic cell. Ser-65

Tyr-66

Gly-67

OH

OH

238 aa precursor

O CH2O O H N C C N C C N C C H H H H H2 CH2

folding τ = 10 min

OH

OH

cyclisation

CH2

CH2

O C HN H C O H N C C N C C H H2 CH O

O C HN H C O H N C C N C C H H2 CH OH

2

2

OH

OH τ = 3 min dehydration

H2O

OH

OH

Aequorea victoria GFP

O2

H2O2

CH2

CH

λex = 395 + 488 nm, λem = 508 nm N

C

C

O H N C C N C C H H2 CH2 OH

O C N H C O

O

oxidation τ~1h

H N C C N C C H H2 CH2 OH

The reaction increases the system of conjugated double bonds (π-system) compared to Tyr and shifts the absorbtion maximum from 280 to 395 nm.

Stereo representation of the crystal structure of GFP (PDB-code 1ema) The protein consists mainly of antiparallel β-strands, which together form a β-barrel. An α-helix with the fluorophore runs in the center of the barrel, where the fluorophore is protected from collisions with water and, in particular, oxygen. Any such collision would prevent fluorescence by taking away energy from the excited fluorophore. This accounts for the high

64

Protein structure

3.2.3

quantum efficiency (green photons produced per blue photons absorbed) of GFP.

The amino acids that interact with the fluorophore in the active center of GFP GFP has 2 absorption maxima, that of the non-ionized fluorophore (phenol) at 395 nm (UV) and that of the ionized (phenolate) at 488 nm (blue).

In the native protein Ser-65 donates a hydrogen bond to Glu-222 which makes deprotonation of Glu-222 easier. The negative charge on this residue then prevents deprotonation of the phenyl-group of the fluorophore. If Ser-65 is mutated to Ala, ionization of the fluorophore becomes easier, the absorbtion maximum at 395 nm is reduced and that at 488 nm becomes stronger. The phenolate ion forms a hydrogen bond with Thr-203, if this is mutated to Ile the fluorophore is stabilized in the phenol-form and the absorbtion maximum at 395 nm becomes stronger at the expense of that at 488. If Thr-203 is mutated to an aromatic amino acid like Tyr stacking of the π-systems leads to a red-shift of both absorbtion and emission maxima by 20 nm because of the reduced exited state energy (yellow fluorescent protein). If Tyr-66 is replaced by Trp or His, the maxima are blue-shifted to 436/476 nm (cyan fluorescent protein) and 390/450 nm (blue fluorescent protein), respectively.

65

3.2.3

Biochemistry and Genetics

Figure 3.24.: Biuret reaction of proteins. The Cu2+ is reduced by the protein in alkaline solution to Cu+ , which forms a purple complex with the protein. The Cu+ can undergo further reactions, which are the basis of the BCA and Lowry-tests. Figure taken from [Buxbaum, in press]. R O N H

C C

H N

R O

R O

H

C C

C C

N

-

OH

H2O R O N H

C C

_ N

R O C C

R O

H

C C

N

2+

Cu

+

C C O R

N H

R O

R O

C C

C C

N

Cu C C O R

N H

+

C C O R

H N N H

N H

C C O R

N H

C C

H N

O R

R O C C

C C

H N

O R

purple complex λmax = 540 nm

3.2.3. Proteins in the laboratory Determination of protein concentration Absorbance Proteins do not absorb visible light (380–760 nm) and are uncolored unless they contain a colored prosthetic group (flavins, heme, Cu, Fe...). However, almost all proteins contain tyrosine and/or tryptophan. The aromatic rings of these amino acids absorb UV-light, with a maximum at 280 nm. The biuret assay In alkaline solution, copper salts (Cu2+ ) are reduced by the protein to Cu+ , which forms a violet complex with substances containing two or more peptide bonds (see fig. 3.24 The Lowry Method Similar to biuret, but more sensitive. The Cu+ produced in the biuret reaction and the tyrosine residues in the protein react with molybdophosphoric acid

66

3.2.3

Proteins in the laboratory

Figure 3.25.: Reaction of amino acids and other primary amines with ninhydrin. Note that Pro is a secondary amine and gives a different reaction, turning yellow instead of purple. O

COO OH

2 OH

H3N

+

+

-

CH R

O Ninhydrin

O

O

+

N O

O

O

CH

+

CO2

+

H3O+

R

Ruhemann's purple (λmax = 570 nm)

(Mo6+ ), forming molybdenum blue (Mo4+ and Mo5+ ). Very commonly used, but timeconsuming. Color yield depends on the Tyr-content of the protein. Interference by complex forming and reducing agents, detergents and many other common chemicals. BCA-reaction The Cu+ formed in the biuret reaction forms an intensively purple complex with bichinchonic acid (BCA). Interference by complex-forming and reducing chemicals, but not by detergents. The ninhydrin reaction Ninhydrin reacts with primary amino groups to yield a purple product. Used for free amino acids (see fig. 3.25), also proteins after hydrolysis. Fluorescent amine reagents like OPA and fluoram react with primary amino groups in proteins. Since most labs don’t have a fluorimeter this type of assay is rarely used. Fluorescent yield depends on aa composition of the protein. Bradford-assay This method is based on the fact that proteins bind hydrophobic dyes like CBB-G250, which have different colors in aqueous and hydrophobic environment. Color yield depends on the properties of the protein.

67

3.2.3

Biochemistry and Genetics

Figure 3.26.: Proteins are easily damaged when not handled with care. Just as in medicine the first rule is “Firstly, do no harm”. Figure taken from the Pierce-catalogue.

Separation and purification of proteins The purification of proteins is the first step in elucidating their properties, since in a mixture you could never tell which protein is responsible for an observed effect. Purification is usually not possible with a single technique, but requires the judicious combination of several steps. Since we can not really predict the behavior of a protein in the various techniques, protein purification still is more art than science and requires experienced operators.

Crude separation Precipitation Proteins can be precipitated without denaturing them by Adjustment of pH Protein solubility is minimal at the isoelectric point. Adjustment of salt concentration A modest salt content enhances solubility because intermolecular salt bonds are disrupted. High salt concentrations (> 10 %) reduce the solubility because the salt competes with the protein for water. Organic solvents The addition of water-miscible organic solvents (ethanol , acetone ) at low temperature precipitates proteins, usually without denaturing them. Dialysis This method uses a porous cellophane membrane to separate molecules by size: salt and small molecules are removed from the protein solution.

68

3.2.3

Proteins in the laboratory

Figure 3.27.: Globulins are, as the name implies, spherical molecules with an even charge distribution. Such proteins are soluble in distilled water and low concentration salt solutions, they are precipitated by high salt concentrations. Albumins on the other hand have an asymmetric shape and charge distribution, they are held together by ionic bonds. As a consequence they are not soluble in distilled water, low salt concentrations are required to break these bonds (salting in). High concentrations of salt precipitate the protein again. Figure taken from [Buxbaum, 2007]. Globulins _+ +_

_ + _

+_

+

Albumins +_

+

+

+ _ _+

_

+

+

_ + _

+_

+

_ + _

+_

_+

+ _ _+

+_ +_

+

+ _

+

+ _

+ _ _+

+

+

+ _

_ + _ _

+ +

+

_

_

_ +

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+

+ _ _

+

_ + _

_

_

_

_

+ +

+ _

+ + _

_

+

_

_ +

_

+

+

_

+ +_

_

+

_

_

_

+

+

_

+

+ _+ +_

_

_

_ +

_ +

_

_ _

+ +

+

_

_

Figure 3.28.: Principle of chromatography. For details see text. Figure taken from [Buxbaum, 2007]. 6ROYHQW

0DWUL[

7HVWWXEH ZLWKIUDFWLRQ

WLPH

 







      

ion exchange

affinity, HIC

gel filtration

69

3.2.3

Biochemistry and Genetics

Chromatography Chromatography is a widely used technique to separate mixtures of different compounds either on an analytical or a preparative scale. The basic principle is that compounds partition between a stationary (usually solid, sometimes liquid) and a mobile (fluid or gas) phase. The partitioning coefficient Kp = [A]m /[A]s determines how fast the substance A moves. From the various chromatographic formats only column chromatography is used to separate proteins. Gel filtration (size exclusion chromatography (SEC)) This method separates proteins (and other macromolecules) by molecular size using small porous beads of a cross-linked gel in a column chromatographic procedure. Big molecules come out first. Ion exchange chromatography (IEC) The stationary phase contains charged groups which interact with oppositely charged groups on the protein. Proteins are eluted by increasing the salt concentration and/or changing the pH. Affinity chromatography A ligand (substrate, antibody, inhibitor, etc.) is covalently linked to the stationary phase. The target protein binds and is then eluted with free ligand. Reversed phase Chromatography (RPC) A lab work-horse for the separation of drugmolecules, amino acids, peptides and small proteins. This method normally separates based on the selective retention of analytes on a hydrophobic ligand (usually C18 ). An organic solvent like acetonitrile is used to chase off the retained proteins one at a time. This method would denature proteins, so much less hydrophobic residues are used (butyl- or phenyl-): hydrophobic interaction chromatography (HIC). Proteins are loaded onto the column at high salt concentrations to maximize their interactions with the hydrophobic groups, elution is with a gradient of decreasing salt concentrations. Electrophoresis Proteins are charged if the pH6= pI. Thus they can be separated in an electric field, depending on the method used separation can be on size+shape, charge or pI. Since electrical currents produce heat, which denatures proteins, electrophoretic methods are usually used for small-scale (analytical) separations, where the large surface area / volume ratio makes cooling easier. Electrophoresis At a pH above its isoelectric point, a protein migrates to the anode, below the isoelectric point to the cathode. Electrophoresis can be done in gels, on thin layer, cellulose acetate foils, etc. It is used in clinical laboratories to separate plasma proteins or diagnostically important isoenzymes. It separates molecules by their charge/mass ratio. However, in the presence of a charged detergent like sodium dodecylsulphate (SDS) or cetyl trimetylammonium bromide (CTAB) proteins bind a roughly constant amount of detergent (1 detergent molecule per 3 amino acids), the charge of the bound detergent is then much larger than that of the protein itself. Hence all proteins have the same charge/mass ratio and hence experience the same acceleration in an

70

3.2.3

Proteins in the laboratory





 







 





















Mw (kDa)

Figure 3.29.: Separation of proteins by size in polyacrylamide gel electrophorese (PAGE). Figure taken from [Buxbaum, 2007].

























200 



































100



























50















Myosin (200) Galactosidase (116.3) Phosphorylase b (97.4) Bovine serum albumine (66.2) Ovalbumine (45)



 



Carbonic anhydrase (31)

 

























20 























Trypsin inhibitor (21.5) Lysozyme (14.4)

10

Gel

add sample

separate

relative migration distance

Figure 3.30.: isoelectric focussing (IEF) separates proteins by pI. Figure taken from [Buxbaum, 2007]

71

3.2.3

Biochemistry and Genetics

Figure 3.31.: 2D-electrophoresis of proteins. For details see text. Figure taken from [Buxbaum, 2007].

pH 3

pH 10

200 kDa

14 kDa mount IEF gel and load molecular mass marker

after running 2nd dimension

electric field. However, their retention by a network of cross-linked gel-molecules is size dependent. Isoelectric focusing IEF a pH gradient is set up in a gel. In the electrical field, proteins stop migrating when the local pH equals their pI. 2D-electrophoresis Proteins from a sample are first separated by pI using IEF, then in a second run by size using SDS-PAGE. If this is done carefully, about 10 000 protein spots can be resolved from a cell extract. This is a key method of proteomics, where the protein content of healthy and diseased cells are compared to identify possible drug targets.

Membrane proteins Membrane proteins are more difficult to purify than soluble ones, because you first have to get them out of the membrane. Membrane attached proteins can be washed of the membrane by high salt concentrations or high pH, but proteins with transmembrane segments have to be solubilized with detergents. Detergents are molecules with a hydrophilic and a hydrophobic end, that is, they are amphophilic. For this reason they can mediate between aqueous and lipophilic phases. The use of detergents for cleaning makes use of this effect: The hydrophobic tails of the detergent insert into an oil droplet, the hydrophilic head groups allow the complex to stay in the aqueous phase. In addition, many detergents bear a charge on their head groups, Coulombic repulsion between the head groups cause the oil droplets to disperse.

72

Proteins in the laboratory

3.2.3

Figure 3.32.: Left: Detergent look a little bit like phospholipids, with a hydrophobic tail and a hydrophilic head group. The head group may be uncharged, or it may bear positive or negative charges. Right: Detergents tend to aggregate in aqueous solution into micelles, where the hydrophobic tails point into the interior and are shielded from the water. Figures taken from [Buxbaum, 2007]. O H2 O C O CH Phosphatidylcholine

O

C O P O H2 O

O

CH3 +

C C N CH3 H2 H2 CH3

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1111 0000 0000 1111 0000 1111 0000 1111 0000 1111

11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00

1111 0000 0000 1111 0000 1111 0000 1111

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11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00 11 00

Figure 3.33.: Solubilization of membrane proteins by detergents. The detergents form torroidal micelles around the transmembrane section of the proteins. In the micelles the hydrophobic tails of the detergent interacts with the hydrophobic amino acid side chains, the hydrophilic head groups keep the micelle in solution. Each micelle contains only a single protein molecule, making purification possible. Once the protein is pure, it can be re-inserted into an artificial lipid membrane, a liposome for further study. Figure taken from [Buxbaum, 2007].

73

3.3

Biochemistry and Genetics

Protein structural analysis Amino acids sequences are known for many proteins. Steps in primary structural analysis include: Amino acid analysis Complete acid hydrolysis of the protein, followed by HPLC separation of the amino acids for quantitative analysis. Fragmentation of the protein into shorter peptides usually with proteolytic enzymes. The fragments may be analyzed by mass spectrometry. Amino-terminus determination using chemicals which selectively react with the terminal amino group. Sequencing using Edman degradation chemistry. Nowadays proteins sequences are often determined indirectly by sequencing their genes, which is a lot less time consuming. Secondary structure analysis is usually done using biophysical methods like circular dichroism or infrared spectroscopy, which measure the α-helix and β-sheet content. Tertiary and quaternary structure analysis are now done using X-ray crystal structure determination and nuclear magnetic resonance (NMR) spectroscopy. Verification of structures at least of peptides and small proteins is done by synthesizing them from amino acids by the Merrifield-method.

3.3. Protein folding diseases So far we have looked upon protein folding as driven by the reduction of ∆G to one stable, called native, structure. However, there are proteins that have a metastable native structure and that can fold into one or even several more stable structures with different biological properties. Often, these proteins contain large segments of coil, with little or no other secondary structure (intrinsically disordered proteins). When these segments come into contact with a template protein they refold to fit that template, with the free energy of binding driving the process. We have discussed above how this can be useful for regulatory proteins. The problem is that contact of some proteins with their alternative conformation results in their autocatalytic conversion into the alternative conformation, which is not only devoid of the biological function but also has a tendency to form aggregates, called amyloid. Surprisingly all amyloid seem to have basically the same structure, fibres made of β-helices. Many proteins that tend to form amyloid tend to have more Gln and Asn, aromatic and β-branched chain and fewer polar amino acids than average proteins, however, this is not a universal feature.

74

3.3

Protein folding diseases

Figure 3.34.: Edman-degradation of proteins. The amino acids are cleaved of one by one from the N-terminus and analyzed. After each cycle the amino group of the next amino acid is exposed for cleavage. About 50 amino acids can be sequenced this way, longer proteins must be cut into segments first. Figure taken from [Buxbaum, 2007]. H NH2 R CH C O R'

N C S Phenylisothiocyanate

N C S NH R CH

NH

C O

CH

NH

C O

R'

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Protein Phenylthiourea

HCl

NH2 O C H C R

N

R' C S

+

CH C O

NH

Phenylthiohydantoin amino acid

Protein (with N-terminal aa removed)

75

3.3

Biochemistry and Genetics

Figure 3.35.: Merrifield-synthesis of peptides. An amino acid, with its amino-group protected by a Fmoc-group, is bound to a solid support via it’s carboxy-group. The protective Fmoc-group is cleaved away so that it can react with a second amino acid. The amino-group of that amino acid is protected by Fmoc, the carboxy-group activated by DCCD to make the reaction with the exposed amino-group of the first amino acid easier. This cycle of deprotection and coupling is repeated until the peptide is fully synthesized. Then the peptide is cleaved of the support and purified. Proteins of up to 100 amino acids can be synthesized this way. Important: Unlike biological protein synthesis, which goes from the N- to the C-terminus, chemical synthesis goes from C- to N-. You need to keep that in mind when talking about sequences to a chemist! Figure taken from [Buxbaum, in press]. OH O C H N CH

fmoc

fmoc

HCl

R1

O

C H N CH

fmoc C H2

H2C

O

O

+ Cl

Polystyrene

H2C

mild organic base

H2N CH

R1

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C

Polystyrene

O

R1

fmoc

DCCD O C H N CH R1 DCU

O

fmoc =

C O

DCCD =

CH2

N H C

DCU =

N

NH C O

O

NH

O fmoc

H2C O

Polystyrene

C

H N CH C

H N CH

R1

R1 mild organic base

F C H2 O O O

H2C

C

R1

O O

C

N CH H R1 N CH H R1 C

Polystyrene

fmoc

O O C H N CH

HF

H N CH

CH C N H R1 N CH H R1

76

OH C

Polystyrene

O

R1 O

H2C O

C

H N CH C

H2N CH R1

R1

Polystyrene

Spongiform encephalopathies

3.3.1

Figure 3.36.: Structure of amyloid formed by a yeast prion protein (HET-s fragment 218289, PDB-code 2rnm), determined by solid state (magic angle spinning) NMR. Each protein forms a β-helix of 2 turns with 3 strands each. The helices come together from different proteins (represented by different colors) to form extended parallel β-sheet.

In yeasts there are several such proteins; the amyloid states are transferred to the daughter cells in mitosis and meiosis (dominant), inheritance is in a non-Mendelian, cytosolic manner. Some of these amyloids can be cured by growing cells in the presence of guanidinium hydrochloride (GuHCl), a compound which unfolds proteins. Low temperature also prevent the autocatalytic amyloid formation and can even break up amyloid fibrils; apparently amyloid formation is driven mainly by entropy. Amyloids are involved in – many say: the cause of – several severe human diseases. It is however unclear whether the pathogenesis is really caused by the large amyloid aggregates or rather by soluble, oligomeric intermediates. The latter have been shown to solubilize phospholipid vesicles. In addition, trapping of other proteins in the aggregate – which then can not perform their normal function – may also play a role. Because the conversion of proteins into amyloid is a slow process amyloidoses tend to manifest in the brain, where cell turnover is low, at advanced age and in the form of a slow degeneration. In our aging society this puts severe strain on public health budgets and the families of the affected. Because disease onset is past the usual age of reproduction there is little evolutionary pressure against these diseases. Detection of amyloid in histologic sections often relies on the metachromatic shift of color or fluorescence of dyes bound to them, examples include Congo Red and Thioflavin T. The effect is enhanced when the sample is viewed between crossed polarizers.

3.3.1. Spongiform encephalopathies The first spongiform encephalopathy known was scrapie in sheep, first described in the 18th century. Other diseases affect humans:

77

3.3.1

Biochemistry and Genetics

Figure 3.37.: Left: X-ray structure of part of the normal PrPc . Middle: Hypothetical model of PrPsc . Figure taken from [Doenecke et al., 2005]. Right: Vacuolization in the brain of a patient with CJD. Figure curtesy of Dr. Yakubovskyy, RUSM.

Gerstmann-Stäussler-Scheinker-disease (GSS) Patients have difficulty to coordinate their movement (ataxia, nystagmus, tremor), loose their speech and finally control over body functions. Disease is protracted (1–11 a). Familiar cases have been described with F198S or P102L in the prion protein. Creutzfeldt-Jakob-disease (CJD) Patients suffer from myoclonus, ataxia, hallucinations, loss of memory, change of personality and dementia, in the final stages akinetic mutism (decerebrate rigidity). Death usually occurs within 4–6 mo after first symptoms. In familial cases the patients are younger than in sporadic (40 vs 65 a), mutations E200K and V210I in prion protein have been reported. fatal familial insomnia (FFI) At age 40–50 patients suddenly fail to sleep. Sleeping pills show no effect. Secondary to sleep deprivation dysautonomia develops: myosis, elevated blood pressure, heart rate and temperature, profuse sweating, myoclonus, impotency. As the disease progresses, patients loose the ability to walk, keep their balance, control their sphincter, speak. Hallucinations, panick attacks, agitation and phobias develop, but unlike the other prion diseases patients retain their mental capacity until almost the end. Death occurs between 0.5 and 3 a after onset. On autopsy one finds neuronal degradation and reactive astrocytosis in the anterior and dorsomedial thalamic nuclei, but without spongiosis. The disease is usually familial with D178N of the prion protein. However, FFI occurs only if amino acid 129 is Met, if it is Val, the D178N mutation leads to CJD. Spontaneous cases of FFI have also been reported.

78

Spongiform encephalopathies

3.3.1

Kuru is a disease spread by endo-cannibalism (ritual eating of deceased family members) in the Fore-people in Papua-New Guinea. Although this custom has been stomped out by the colonial power (Australia) in the 1950s, occasional new cases of kuru are reported in elderly patients due to the extremely long incubation period of the disease. Patients giggle and tremble uncontrollably (kuru = the laughing death), then loose control over body and mind and finally die after about one year. variant Creutzfeldt-Jakob-disease (vCJD) or “mad cow disease”, similar to kuru, is spread by ingestion of infected meat, but the meat of British cows that during the 1980s had been fed with offal from scrapie-infected sheep without adequate precautions. Prions which have crossed the species-barrier once are apparently much more likely to do so again, so unlike scrapie vCJD can be spread to humans. The agent crosses into the gut associated lymphatics and from there moves up the neural tissues via spinal cord into the brain. Distinction between classical and variant CJD can be made by tonsil biopsy, only in vCJD will it contain prions. Compared to classical CJD patients are younger (median 29 instead of 65 a), the course of the disease is more protracted (14 vs 4.5 mo) and the first symptoms are usually psychiatric (depression, aggression and loss of memory). About 170 patients have been reported, 152 in Britain alone. Of particular concern are iatrogenic transmissions of spongiform encephalopathy by surgical instruments, neural tissue (Dura mater or corneas), or pharmaceuticals made from brain (e.g. growth hormone, gonadotropin). No transmissions by blood transfusion have been described so far, but it is considered a probable route. All these diseases have in common the development of a spongiform encephalopathy, where vacuoles form in the brain at disease-specific sites (see fig. 3.37, right). The diseases can occur sporadically or familial, but they can also be transmitted by ingestion of tissue from affected individuals. Unusual in this context is the extreme stability of the pathogenic agent against decomposition, heat, radiation or disinfectants. The brain of a sheep with scrapie, when fed to mice, proved infective after burial for 3 a! T. Alper and her co-workers noticed in the 1960s that the agent is not destroyed by UV-light of 250 nm or by nucleases, she therefore concluded that the agent may consist of proteins only. S. Prusiner has vigorously followed up on this heretic idea and managed to identify the protein involved (N.P. 1997), called prion protein (PrP). Prion stands for “proteinaceous infectious agent”. PrP is a membrane protein of unknown function (knock-out mice are phenotypically normal, except that they do not get spongiform encephalopathy after ingesting scrapie-infected brains). In normal brain PrP has a secondary structure consisting mostly of α-helices (PrPc , c = cellular, see fig. 3.37, left). In spongiform encephalopathy this protein changes its secondary structure into a β-pleated sheet, this leads to protein aggregation (PrPsc , sc = scrapie (see fig. 3.37, middle). The aggregates form long fibrils (see fig. 3.36). It is not known

79

3.3.2

Biochemistry and Genetics

whether the aggregates themselves are cytotoxic, or whether they are inert and the damage is caused by a soluble intermediate. The key point for understanding of prion diseases however is the realization that the conversion from PrPc to PrPsc is auto-catalytic, in other words: a molecule of PrPc that comes into contact with PrPsc changes its conformation into PrPsc . It gets even weirder than that: There exists not only one alternative conformation of PrP, but the different spongiform encephalopathies are caused by different strains of PrPsc , which have different conformations. These different conformations can be differentiated by their sensitivity to proteases or chemicals that break up protein secondary structure like guanidinium hydrochloride or urea.

3.3.2. Morbus Alzheimer (dementia) is named after Alois Alzheimer (German psychiatrist and neuropathologist, 1864–1915), who first described it in 1906. The disease is clinically characterized by the loss of (short term) memory, excitation, apathy, paranoia, depression, aggression with possible violence, progressing over loss of language, immobility, incontinence to finally death. Usual age of onset is ≥ 65 a, this disease is spontaneous and age related. About 2 % of the population at age 65 a is affected, by age 80 a prevalence increases to 20 %. Beyond age 85 a the prevalence decreases again, as the patients rarely reach that age. With the growing number of elderly in advanced societies these patients already put a considerable strain on their families and the public health system, this is likely to increase in future. WHO estimates that there are currently about 29 × 106 patients living with Alzheimer’s disease, and that this number will increase to 106 × 106 in 2050 (when the incidence will be 1:85). There is also an inherited form of the disease characterized by an early onset (≤ 60 a). Strictly speaking, the case described by Alzheimer was early-onset (pre-senile dementia), the late onset form should be called “senile dementia of the Alzheimer type (SDAT)”, but this distinction is rarely made. In Alzheimer’s disease the β-amyloid precursor protein (APP) is proteolytically cleaved extracellularly by β-secretase, then within the membrane by γ-secretase. The extracellular fragment resulting from the latter cleavage is the β-amyloid, which forms neuritic plaques. If instead by β-secretase the APP is first cleaved by α-secretase then the product resulting from γ-secretase cleavage can not form plaques. The cytosolic part of APP left over after cleavage is called APP intracellular domain (AICD). The role of AICD in Alzheimer’s disease is controversial, as it appears short-lived. However, it interacts with about 20 identified partner proteins. Amongst those is the histone acetyltransferase TIP60 (affects DNA/histone interactions and hence gene transcription) and the adapter FE65 (increases stability of AICD). The AICD/FE65/TIP60 complex (“AFT”) can be found in the nucleus, where it may act as transcription factor. In addition to the extracellular neuritic

80

3.3.2

Morbus Alzheimer

soluble APP

Figure 3.38.: Amyloid formation in Alzheimer’s disease.

extracellular membrane

Secretase

Amyloid

Secretase

aggregate

AICD

intracellular

Secretase

amyloid precursor protein

Secretase

Amyloid

NH 2

COOH

Figure 3.39.: positron emission tomography (PET) scan of the brains of left: normal 20 a old, middle: normal 80 a old, right: 80 a old with Alzheimer’s disease. Colors denote metabolic activity in the brain (red = high to blue = low). Figure © Alzheimer’s Disease Education and Referral Center, National Institute on Aging

plaques, histology of brains from patients with Alzheimer’s disease also reveals neurofibrillary tangles inside the cells. These consist of hyperphosphorylated τ-protein (component of the cell skeleton). One of the potential target genes identified for AFT is glycogen synthase kinase 3β, which is one of the kinases responsible for τ-phosphorylation. If these results could be confirmed, then AFT would connect plaque and tangle formation. Further investigation into the biochemistry of Alzheimer-brains reveals dysfunctional mitochondria which produce high amounts of reactive oxygen species (ROS) (more about ROS in the lecture on oxidative phosphorylation, see chapter 15.3 on page 253). ROS are very toxic to cells. It is currently unclear what the patho-mechanistic relationship between plaques, tangles and ROS is. In early onset (familial) Alzheimer’s disease several mutations have been found. The genes PSEN1 (AD3, on chromosome 14) and PSEN2 (AD4, on chromosome 1) code for

81

3.3.3

Biochemistry and Genetics

Figure 3.40.: Left: neuritic plaques (amyloid) and neurofibrillary tangle (hyperphosphorylated τ-protein) in the brain of a patient with Alzheimer’s disease. Right: amyloid angiopathy, resulting in brain hemorrhage. Pictures curtesy of Dr. Yakubovskyy, Dept. of Pathology, RUSM.

presenilin 1 and 2, respectively, which are components of γ-secretase. Mutations in APP (AD1, on chromosome 21q) have also been found. There is also significant association between Alzheimer’s disease and the 4 allele of the ApoE protein (AD2, on chromosome 19), which is involved in the transport of cholesterol in blood. In addition, mitochondrial DNA-polymorphism and several other mutations have been described as associated with Alzheimer’s disease. Patients with Down’s syndrome (trisomy 21) are at increased risk for Alzheimer’s too, because of their general mental deficiency the onset of Alzheimer’s is particularly difficult to diagnose in these patients. Because the cause of Alzheimer’s disease is unknown, prevention is in infancy. The following recommendations however are widely agreed upon: • control of blood pressure and [cholesterol] • balanced diet with minerals and vitamins • no smoking • profession with high intellectual activity • high physical activity • limited TV consumption Several authors have described a connection between Alzheimer’s disease and high aluminium ion intake (e.g. from cooking utensils), however, this is now considered a red herring. Treatment of Alzheimer’s disease is possible with various pharmaceuticals that have come onto the market in the last couple of years, but all of them only reduce the symptoms, they do not slow down disease progression and do not change the final outcome.

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Chorea Huntington

3.3.4

3.3.3. Morbus Parkinson The first description of this disease in modern medical literature was by the English physician James Parkinson in 1817, but several ancient sources describe what appears to be Parkinson’s disease, e.g. in China the Yellow Emperor’s Internal Classics from 425 BC and the Ayurveda in India (≈ 1000 BC). In Parkinson’s disease (shaking palsy) the protein α-synuclein aggregates in the substantia nigra of the brain, forming Lewy-bodies. This leads to a failure of ER → Golgi transport, resulting in the death of extrapyramidal cells in the pars compacta of the substantia nigra. These cells would normally produce dopamine which acts on basal ganglia. This results in a characteristic trembling, in slow movement and finally the cessation of movement. Patients show a characteristic, bend-forward posture when standing. Hallucinations, depression and other psychiatric symptoms may be seen. The disease usually strikes between 50 and 60 a of age, more often in ♂ than in ♀. Morbus Parkinson is usually caused by gene duplication, but contact with toxic chemicals (pesticides, trichloroethylene) can show similar results. Precursors of dopamine (L-Dopa (Levodopa)), dopamine agonists or substances that interfere with dopamine breakdown (MAO-B or COMT inhibitors) are used to treat the disease. You will learn to understand their action in the next semester. It is fascinating that the herbal remedies described in ancient Chinese sources contain substances that act like modern drugs against this disease.

3.3.4. Chorea Huntington Huntington’s disease is caused by an expansion of CAG-repeats (base triplet encoding for Gln, see the genetic code in the appendix) from 10–35 in the protein huntingtin. Poly-Gln > 40 amino acids form β-sheets which lead to aggregation. Inheritance is dominant autosomal with complete penetrance, chromosome 4p16.3. Huntingtin is required for endocytosis and hence for recycling of vesicle membranes after exocytosis. The death of brain cells in basal ganglia (putamen + caudate nucleus) reduces indirect inhibition of globus pallidus internus, resulting in activation of thalamus and cortex. Huntington’s disease is characterized by jerky movements (choreoathetosis, choreia = Gr. dance), cognitive and behavioral defects. There are several other trinucleotide expansion diseases in other proteins. Not fully understood is the role of tissue transglutaminases in Huntington’s and also Parkinson’s disease. Transglutaminases form isopeptide bonds between glutamine (R) and lysine (R’) residues in proteins (R−CO−NH2 + H2 N−R0 → R−CO−NH−R0 + NH3 ), the most well known transglutaminase is factor VIII of the blood clotting cascade (see section 11.1.2 on page 213). Tissue transglutaminase (tTG) is an enzyme found in all organs, including brain. It has been found that intramolecular crosslinks in α-synuclein and tTG

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binding to synuclein increase as the disease progresses, but this may actually be a protective mechanism to reduce amyloid production by preventing the β-sheet formation.

3.4. Example questions 1) Effect of pH on enzyme activity Lysozyme is an enzyme that occurs in tears. It hydrolyzes the murein sacculus of bacteria and thus protects us from eye infection. It’s catalytic center contains two essential, acidic amino acid residues, Glu-35 (pKa = 5.9) and Asp-52 (pKa = 4.5). Their R-groups have to be in the correct state (protonated (−COOH) / deprotonated (−COO− )) for the enzyme to work. The diagram shows measurements of the enzymes activity at constant enzyme and substrate concentration, but different pH. What is the required state of the two R-groups in the active site? pH dependency of lysozyme activity 100 90

Activity (% of maximum)

80 70 60 50 40 30 20 10 0

2

3

4

5

6 pH

7

A Glu-35 protonated, Asp-52 deprotonated B Glu-35 protonated, Asp-52 protonated C Glu-35 deprotonated, Asp-52 deprotonated D Glu-35 deprotonated, Asp-52 protonated E it does not matter

84

8

9

10

3.4

Example questions

2) Structure of alpha-amino acids, radioactivity Which of the following statements is false? Alanine, labeled with 14 C (β-emitter, half life period = 5500 a) on the carboxygroup, A produces electrons by radioactive decay. B when decarboxylated produces radioactive CO2 and non-radioactive ethylamine. C may be produced by sparging a solution of non-radioactive alanine with

14 CO

2.

D has the same pI as non-radioactive alanine. E can be used by cells to make proteins.

3) pI-value of amino acid Lysine has the pKa -values 2.18 (−COOH), 8.95 (α-amino) and 10.53 (-amino). What is the pI? A 3.50 B 5.57 C 7.22 D 9.74 E 11.83

4) Properties of amino acids Sickle cell anemia, an inherited disease, is caused by the mutation Glu6Val in the β-subunit of hemoglobin. Compared to the normal protein you would expect the mutated protein to move in a native electrophoresis experiment (that is, without SDS): A) less to (+) at pH 8, same distance at pH 1 B) same at pH 8, more to the positive at pH 1 C) more to (+) pH 8, more to the (-) at pH 1 D) less to the (-) at pH 8, less to the (-) pH 1 E) same distance under all conditions

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5) Functional replacement of amino acids You are working on a research project to elucidate the reaction mechanism of an enzyme. You think that a particular serine residue in the protein is required for catalytic activity. To test this hypothesis you want to genetically replace this amino acid by another, and then test whether the enzyme is still active. Which amino acid would you choose to replace the Ser? A Threonine B Alanine C Tryptophan D Glutamic acid E Histidine 6) pH dependence of solubility of amino acids Which of the following tripeptides would you expect to be the most soluble in 1 M NaOH: A) Phe-Ala-Val B) Glu-Gly-Asp C) Gln-Gly-Asn D) Lys-Arg-His E) Trp-Lys-Asn 7) Determination of protein concentration from UV-absorption Blood serum contains many different proteins at a fairly constant concentration (6.0–7.8 g/dl). However, in several serious diseases protein concentration is lowered (e.g. liver cirrhosis) or elevated (e.g. multiple myeloma). A quick way to determine the serum protein concentration is to measure the UV-absorbance at 280 nm. A 1:100 dilution of serum gives an absorbance of 0.4 at 280 nm in a standard cuvette of 1 cm path length. Assume a molar extinction coefficient of 4 × 104 l mol−1 cm−1 and an average molecular weight of 65 kDa for blood proteins. The protein concentration is approximately A 6.0 g/dl B 6.5 g/dl C 7.0 g/dl D 7.5 g/dl E 8.0 g/dl

86

Example questions

3.4

8) Strange disease A.B., 45 a old ♂, visits you because he can not sleep. Hypnotics are without effect. You notice the following signs: myosis, hypertension, tachycardia and elevated body temperature with diaphoresis. Over the following months the patient develops dream-like states, dysarthria, myoclonus and impotency. He looses the ability to walk, keep his balance and control his sphincter. However, his ability to think and understand does not diminish until after 15 m the patient falls into a coma and finally dies quite suddenly. Autopsy shows neuronal degradation with reactive astrocytosis limited to the anterior and dorsomedial thalamic nuclei without spongiosis or inflammation. What is the most likely diagnosis? A astrocytoma B myasthenia gravis C morbus Alzheimer D fatal familial insomnia E new variant Creutzfeld-Jakob disease 9) Collagen related inherited disease X.Y. has malformation of his limbs because his epiphyses ossified from several discrete centers with a stippled appearance in X-ray, the shafts of the long bones are thickened. He also has a congenital cataract of both eyes and mental retardation. Genetic analysis shows a mutation in the gene for collagen α1(II). What is the most likely diagnosis? A chondrodysplasia B Ehlers-Danlos syndrome C osteogenesis imperfecta D Alport syndrome E epidermolysis bullosa 10) CAG-length variation A.B., 45 year old male, visits you in your office because of involuntary movements in arms, legs and face. He is very worried because the (protracted and finally fatal) disease of both his father and paternal grandfather had started with the same symptoms. Upon molecular investigation you find that the repeat-length of a CAGstretch in the gene for a protein involved in endocytosis is 155 (normal up to 35). The most likely diagnosis is: A) Chorea Huntington B) Alzheimer’s disease

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C) Kuru D) Fatal familial insomnia E) Creutzfeldt-Jakob-disease

3.5. Objectives Students should be able to 1. Recognize the structures of the 22 major amino acids. 2. explain how these different structures affect their biological function. 3. explain what pKa -values are and how to calculate the pI. 4. Name the non-covalent interactions that can be formed by the different amino acid side chains. 5. Describe the structure of the peptide bond, including its partial double bond character, planarity and ability to engage in hydrogen bonding. 6. define the terms primary, secondary, tertiary and quaternary structure, fibrous protein, globular protein, albumin and globulin. 7. describe the structure of the α-helix and the β-pleated sheet and the role of hydrogen bonds in their formation. 8. explain on suitable examples how particular structures allow a protein to serve its biological function. 9. name the components of glycoproteins, lipoproteins, nucleoproteins, phosphoproteins, heme proteins, flavoproteins and metalloproteins and describe the interaction between the polypeptide and prosthetic group in each case. 10. describe the process of heat denaturation of proteins and its biological consequences. 11. list various types of denaturing agents and specify the mechanism by which they cause denaturation. 12. state the susceptibilities of peptide bonds and disulfide bonds to acids, bases, oxidizing and reducing agents. 13. know the use of UV absorbance, the biuret and Lowry methods for the measurement of protein concentrations and describe why the result of such measurement depends on the method used.

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Objectives

3.5

14. name the principles by which proteins are separated in different types of chromatography and electrophoresis. 15. describe the post-translational modification of proteins and their interaction with prosthetic groups. 16. explain, using suitable examples, why mutation of a single amino acid in a protein may result in genetic disease. 17. critically discuss the mechanism of prion and other protein folding diseases

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4. DNA and Gene Expression 4.1. DNA Structure 4.1.1. Bases, Nucleosides and Nucleotides DNA contains the two purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and thymine (T). RNA contains uracil (U) instead of thymine. NH2

O

N

N N

N H2N

N H

Adenine

O

Thymine

N H

O CH3

N H

N Guanine

O HN

N

NH2

HN O

N N H Uracil

O

N H Cytosine

Nucleosides consist of a base linked by an N-glycosidic bond to the 1’ -carbon of ribose or 2’-deoxyribose. Examples: Adenosine A + ribose Guanosine G + ribose Cytidine C + ribose Uridine U + ribose 2’-deoxyadenosine A + 2’-deoxyribose 2’-deoxythymidine T + 2’-deoxyribose Nucleotides are nucleoside derivatives carrying 1, 2 or 3 phosphate groups at the 5’-carbon of ribose or 2-deoxyribose. They are named as derivatives of their corresponding nucleosides.

91

4.1.3 Examples: A + ribose + 1 phosphate U + ribose + 3 phosphates

Biochemistry and Genetics

Adenosine monophosphate (AMP) Uridine triphosphate (UTP)

4.1.2. The DNA Double-Helix. DNA is an unbranched polymer of 2-deoxyribonucleoside monophosphates. These nucleotides are linked by phosphodiester bonds between the 3’ end of one 2-deoxyribose and the 5’ end of the next 2-deoxyribose. The phosphate groups are negatively charged. The molecule has a polarity, with a 5’ end and a 3’ end. Conventionally, the 5’ end is written left and the 3’ end right. The primary structure of the DNA strand can be described by its base sequence. The Watson-Crick double helix (= B-DNA) is the principal higher-order structure of DNA. Important features: • The two strands of the double helix are antiparallel. • The bases face inward to the helix axis while the sugar-phosphate backbone forms two ridges. • There are a major groove and a minor groove which are lined by the edges of the bases. Minor grove binding proteins bind to the sugar/phosphate-backbone (e.g. histones), in the major grove proteins can access the bases (e.g. regulators of transcription). • In each strand, the bases are stacked flat one on top of the other. • The bases interact by hydrogen bonds, forming A-T and C-G base pairs • The two strands form a right-handed helix with about 10 bp per turn. This structure has several important implications: • A large number of unique DNA sequences can be generated by permutations of the 4 bases. • The edges of the bases are exposed in the major and minor grooves. Therefore the base sequence of the DNA can be recognized by DNA-binding proteins (see fig. 4.1). • The two strands can be separated easily. • The base sequence of one strand predicts exactly the base sequence of the complementary strand.

92

Supercoiling

4.1.4

Figure 4.1.: Glucocorticoid receptor bound to a glucocorticoid response element DNA (PDB-file 1r40). Major and minor groove of the DNA are clearly visible. The receptor belongs into the class of Zn-finger DNA-binding proteins. It’s 4 Zn-ions are visible, also shown are the 4 Cys-residues that keep each Zn-ion in place. Note however that the Zn-ions do not make contact with the DNA, rather 2 α-helices bind into the major grove of the DNA. The Zn-ions however ensure the correct secondary structure of the protein.

4.1.3. Chemical stability The covalent structure of DNA is quite stable. Rigorous conditions (boiling in strong acid) are required to break the covalent bonds. The double helix, however, is destroyed by heatdenaturation (“melting” of DNA). • Short DNAs melt more easily than long DNAs. • A-T-rich DNA melts more easily than C-G-rich DNA. • Low ionic strength and alkaline pH favor melting. The viscosity of the DNA solution decreases and the UV- absorption (measured at 260 nm) increases. Denatured DNA renatures when cooled slowly. This process is called annealing. Short DNAs anneal much faster than long DNAs.

4.1.4. Supercoiling DNA can be overwound, with less than the canonical 10 base-pairs/turn. This is called a positive supertwist. Or it can be underwound, with more than 10 bp/turn. This is called a negative supertwist. Over- and under-winding can occur when the DNA duplex is circular

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4.2.2

Biochemistry and Genetics

(in prokaryotes), or when the ends of the duplex are firmly attached to structural proteins (in eukaryotes). The supertwisting is regulated by topoisomerases. Usually, the DNA in cells is moderately underwound. This facilitates strand separation for transcription and DNA replication.

4.2. DNA Replication 4.2.1. Semi-conservative replication Mechanism of DNA replication: • The double helix unwinds. The point where unwinding occurs is called the replication fork. • New DNA is synthesized in the replication fork, using the old strand as a template. • The old strand keeps unwinding until the whole DNA is replicated. • The replicated DNA consists of one complete old strand and one complete new strand.

4.2.2. DNA polymerases New DNA is synthesized by DNA polymerases. Important properties of bacterial DNA polymerases. • They require deoxyribonucleoside triphosphate as precursors. dATP, dGTP, dTTP. Pyrophosphate is released during the polymerization reaction. • They synthesize DNA from 5’ → 3’. • They require a single-stranded DNA template. Therefore the parental DNA duplex has to unwind first before the DNA polymerases can start. • They read their template from 3’ → 5’. Therefore the template strand and the new strand are antiparallel. All base-paired nucleic acids are antiparallel. • They incorporate only nucleotides that make proper base pairing with the base in the template strand. • They possess a 3’ -exonuclease activity which is specific for mismatched bases and which is used for proofreading. • Many bacterial DNA polymerases have a 5’ -exonuclease activity which is required for DNA repair.

94

Steps in bacterial DNA replication

4.3.1

• The DNA polymerases require a primer with a free 3’-hydroxy group. Bacteria have three DNA polymerases: Poly I Most important for DNA repair. It has a low binding affinity for its template, and therefore it makes only short pieces of DNA at a time: Low processivity. Poly II Unknown function Poly III The essential enzyme for DNA replication. Very high processivity. Thanks to the 3’-exonuclease activities of the DNA polymerases, the error rate is only about one mis-incorporated base per 1 × 108 nucleotides.

4.2.3. Steps in bacterial DNA replication 1. E. coli has a circular chromosome (≈ 3 × 106 bp) with a single replication origin called Ori-c. Initiator proteins associate with Ori-c to start unwinding. 2. A helicase unwinds the DNA. This requires ATP. 3. The topoisomerase gyrase relaxes positive supertwists and actively pumps negative supertwists into DNA (ATP-dependent). This prevents overwinding ahead of the moving replication fork. The gyrase is not in the replication fork. 4. Single-strand DNA binding proteins (SSB proteins) bind the single-stranded DNA to prevent annealing. 5. A specialized RNA polymerase called primase synthesizes a small RNA primer. 6. Poly III starts DNA synthesis at the 3’ end of the primer. 7. One of the new strands, the leading strand, is synthesized continuously. 8. The other strand, the lagging strand, is synthesized piecemeal. The pieces are called Okazaki fragments. 9. The small RNA pieces at the 5’ ends of the Okazaki fragments are removed by Poly I and replaced by DNA. 10. The fragments of the lagging strand are connected by DNA ligase.

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4.3.3

Biochemistry and Genetics

4.3. RNA and Transcription 4.3.1. RNA Structure RNA can form a double helix like DNA, but is usually single-stranded except in some virus. Cellular RNAs contain both base-paired segments (“stems”) and unpaired segments (“loop”). An RNA strand can also anneal with a complementary DNA strand. Annealing between nucleic acids of different kinds or from different sources, for example DNA with RNA, or human DNA with chimpanzee DNA, is called hybridization.

4.3.2. Types of RNA There are three major types of cellular RNA, all of them synthesized as copies of DNA sequences: Messenger RNA (mRNA) carries the information of the linear DNA sequence to the ribosomes where proteins are synthesized. In eukaryotes each mRNA is copied from a single gene and encodes a single polypeptide chain (“monocistronic” mRNA). In prokaryotes most mRNAs are copied from a string of genes and encode more than one polypeptide (“polycistronic” mRNA). By definition, each gene codes for one polypeptide. Ribosomal RNA (rRNA) is a structural component of the ribosome. There are 3 prokaryotic and 4 eukaryotic rRNAs, each of them present in a single copy in the ribosome. Transfer RNA (tRNA) carries amino acids to the ribosome for protein synthesis.

4.3.3. Transcription RNA synthesis is by transcription from a DNA template. It requires the enzyme RNA polymerase. Mechanism: like DNA synthesis, but • The precursors are ATP, GTP, CTP and UTP. • RNA polymerase does not need a primer • RNA polymerase has no nuclease activities.

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Post-transcriptional processing

4.4.1

Steps in transcription (E. coli ) 1. RNA polymerase, sliding along the DNA, binds to the promoter. This requires the σ (sigma) subunit of RNA polymerase. Promoters look different in different genes, with consensus sequences at about −10 (TATAAT, “Pribnow box”) and −35 bp (TTGACA) from the start of transcription. 2. RNA polymerase, separates a short stretch of DNA duplex. 3. RNA polymerase synthesizes the RNA in the 5’ → 3’ direction using the original strand of the DNA strands as a template strand. The template strand is called the coding strand. The DNA anneals behind the RNA polymerase. 4. There is a termination site at the end of the gene where the RNA polymerase falls off its template. Most bacterial termination sequences contain a palindrome. All bacterial RNAs are synthesized by the same RNA polymerase. The rate-limiting steps in transcription are promoter recognition and strand separation. The error rate of RNA synthesis about 1 in 10 000. The life span of mRNA is only a few minutes in bacteria but several hours in eukaryotes. Only 3 % of the RNA in E. coli is mRNA.

4.3.4. Post-transcriptional processing Modification of RNA after transcription: • mRNA is usually not modified in prokaryotes but translated immediately. But extensive processing occurs in eukaryotes. • tRNA has many modified bases, both in prokaryotes and eukaryotes: methylated bases, pseudouridine, inosine (nucleoside containing hypoxanthine), ribothymidine (thymine bound to ribose) etc. Most tRNAs are processed from precursor transcripts by nuclease cleavages. • rRNA is processed by nucleases from a transcript that contains the sequences of all major ribosomal RNAs (5 S, 16 S, 23 S in bacteria, 5.8 S, 18 S, 28 S in humans). There are some methylated bases (prokaryotes) or ribose residues (eukaryotes) in rRNA.

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4.4.3

Biochemistry and Genetics

4.4. Protein Synthesis 4.4.1. The Genetic Code The genetic code (see fig. 34.4 on page 632) describes the relationship between the base sequence of the mRNA (and the DNA) and the amino acid sequence of the protein. Important features: • A base triplet called a codon on the mRNA specifies an amino acid. • There are 61 amino acid coding codons. One of them (AUG, coding for formyl methionine) is also used as a start codon. There are 3 stop codons: UAA, UAG and UGA. • The code is non-overlapping and comma-less. There is no overlap between codons and no empty spaces in between. • The code is co-linear. The codons on the mRNA are in the same sequence as the encoded amino acids in the polypeptide. • The code is unambiguous. Each codon specifies one and only one amino acid. • The code is degenerate. Most amino acids are specified by more than one codon. • The code is universal. There are only minor deviations, especially in the small genomes of mitochondria and chloroplasts.

4.4.2. tRNA tRNAs are small RNAs (80 nucleotides in length) which bring amino acids to the ribosome. Typical “cloverleaf” structure. The 3’-terminus, ending in the sequence CCA, binds the amino acid through an ester bond with one of the hydroxy groups of ribose. The anticodon base-pairs with the codon of mRNA during translation. There is a certain freedom of pairing between the third codon base and the first anticodon base (“wobble”). Therefore cells can do with less than 61 tRNAs. Aminoacyl-tRNA synthetase 3’ end of the tRNA. ATP is aminoacyl-tRNA synthetase specificity is required for the

98

are cytoplasmic enzymes which transfer an amino acid to the hydrolyzed to AMP + pyrophosphate in this reaction. Each has a high specificity for “its” tRNA and amino acid. This fidelity of the genetic code.

Antibiotics

4.4.5

4.4.3. Ribosomes Ribosomes consist of rRNA + protein. Subunits: 30 S + 50 S = 70 S in bacteria, 40 S + 60 S = 80 S in eukaryotes. The 16 S rRNA (bacteria) or 18 S rRNA (eukaryotes) is in the small subunit, the others in the large subunit. Ribosomes self-assemble from rRNA + ribosomal proteins. The subunits are separate in the resting state and form the complete ribosome only when they synthesize proteins. Magnesium is required for ribosome assembly. The ribosome has 2 binding sites for tRNA: the P site (P = peptidyl) contains the peptidyl-tRNA during the elongation phase, the A site (A = aminoacyl, or acceptor) accepts the newly incoming aminoacyl-tRNA.

4.4.4. Steps in translation 1. The initiation complex is formed when the 30 S subunit binds to the 5’-terminal part of the mRNA and to the initiator-tRNA that carries formyl-methionine (bacteria) or methionine (eukaryotes). In bacteria (but not eukaryotes), the Shine-Delgarno Sequence on the mRNA has to base-pair with a complementary sequence in the 16 S ribosomal RNA. Then the 50 S subunit is added. The formation of the initiation complex requires soluble cytoplasmic proteins called initiation factors, and one GTP is hydrolyzed to GDP + phosphate. 2. The fMet-tRNA is in the P-site, base-paired with the start codon AUG. Elongation starts when an aminoacyl-tRNA is placed into the A site by elongation factor EF-Tu. This requires GTP hydrolysis. 3. Next the peptide bond formed by the peptidyl transferase activity of the large ribosomal subunit. 4. Translocation brings the newly formed peptidyl-tRNA from the A site to the P-site. The mRNA moves 3 nucleotides along the ribosome. This requires GTP hydrolysis. 5. Termination of translation occurs when a stop codon is encountered in the mRNA sequence. The polypeptide is hydrolyzed from the tRNA after the binding of protein releasing factors to the stop colon.

4.4.5. Antibiotics Many (not all) antibiotics inhibit protein synthesis: Rifampicin An inhibitor of bacterial RNA polymerase Actinomycin D Inhibits transcription by DNA-base intercalation.

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Biochemistry and Genetics

Streptomycin Inhibits binding of fMet-tRNA and causes misreading of mRNA in prokaryotes. Tetracycline Inhibits the binding or aminoacyl-tRNA in prokaryotes. Chloramphenicol Inhibits the peptidyl transferase of prokaryotes. Cycloheximide Same as chloramphenicol, but in eukaryotes. Erythromycin Inhibits translocation in prokaryotes. Puromycin A structural analog of aminoacyl-tRNA. Causes premature polypeptide chain termination. Clinical note: Most bacteria can survive for considerable periods of time without protein synthesis. Therefore most drugs inhibiting bacterial protein synthesis are only bacteriostatic, not bactericidal.

4.5. Regulation of Gene Expression Gene expression is regulated, most often at the level of transcription and sometimes by posttranscriptional mechanisms. Proteins that are synthesized sometimes and sometimes not are called inducible. Proteins that are synthesized at all times are called constitutive. Examples in E. coli of gene regulation:

The lac operon contains genes for the catabolism of the disaccharide lactose. Lactose is transported into the cell by the carrier lactose permease and then cleaved into glucose and galactose by the enzyme β-galactosidase. These proteins, together with the enzyme thiogalactoside transacetylase, are produced in the presence of lactose but not its absence. The genes for these 3 proteins are clustered in the lac operon. They produce a polycistronic message which is translated into separate polypeptides. Organization of the lac operon:

100

Regulation of Gene Expression P

I

Repressor mRNA

P

O

A

B

4.5

C

repressor protein binds to operator and blocks transcription of A, B, C genes

Protein

With inducer: Presence of inducer prevents repressor binding to operator

A, B, and C are the genes for β-galactosidase, lactose permease and the transacetylase. These are the structural genes that are expressed as polycistronic unit. P is the promoter, where transcription begins. O is the operator. The operator is a binding site for a repressor protein, next to (and overlapping) the promoter. The binding of the repressor prevents transcription. I is the gene for the repressor. It is expressed constitutively. The repressor binds to the operator in the absence of lactose. In the presence of lactose the repressor binds allo-lactose (formed from lactose). Allo-lactose binding causes a conformational change in the repressor which prevents its binding to the operator, thus allowing RNA polymerase to transcribe the structural genes. The operon consists of a promoter, operator and structural genes. The I gene may be next to the operon (as in the lac operon), but this is not always the case. Other Catabolic Operons are also induced by nutrients, usually by the removal of a repressor protein from the operator. The Tryptophan Operon contains 5 structural genes which encode enzymes for tryptophan biosynthesis. These genes are expressed in the absence but not the presence of external tryptophan. In this case the repressor protein itself (“apo-repressor”) does not bind to the operator, but the binding of tryptophan to the repressor induces a conformational change which causes the repressor to bind to the operator: Tryptophan acts as a corepressor. Tryptophan makes feedback inhibition of its own synthesis. Catabolite Repression is a general regulatory mechanism for carbon metabolism in E. coli. Because glucose is the favored tasty treat of bacteria (it enters glycolysis directly), many catabolic operons in E. coli are transcribed only in the absence of glucose. The level of cyclic AMP (cAMP) is low when glucose is present and high when glucose is absent. cAMP binds to CAP (Catabolite Gene Activator Protein). The CAP-cAMP complex binds

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4.6.2

Biochemistry and Genetics

to the promoter region of many catabolic operons and stimulates RNA polymerase binding and transcription.

4.6. Virus 4.6.1. Virus Structure Virus are obligatory intracellular parasites: they can replicate only by infecting a host cell. Outside the cell, the virus exists as an inert particle called a virion. The virion consists of: Nucleic acid: DNA or RNA, single-stranded or double-stranded, but only one kind is present. Virus have anywhere between 3 and 250 genes. The capsid is a protein capsule that surrounds the nucleic acid. It is formed from a few structural proteins which polymerize into a symmetrical shape. Nucleic acid + capsid are called nucleocapsid. An envelope is present only in some animal virus, not in bacteriophages (=bacteria-infecting virus). It is a piece of membrane acquired from the host cell that contains viral proteins called spike proteins.

4.6.2. The Lytic Cycle of Bacteriophage T4 Lytic infection by the DNA bacteriophage T4 and related phages proceeds in the following sequence: Adsorption: The tips of the tail fibers attach to a surface component of E. coli which serves as a “phage receptor”. This determines the host range: bacteria without the phage receptor cannot be infected. Penetration: the tail sheath contracts, the phage DNA is injected into bacterium. The capsid remains outside. Synthesis of phage proteins: The phage DNA serves as a template for mRNA synthesis, and viral proteins are synthesized on bacterial ribosomes. Phage proteins produced include: - A DNase which degrades the host genome. Viral DNA is protected because it contains hydroxymethylcytosine instead of ordinary cytosine - Enzymes for nucleotide biosynthesis - DNA polymerase, DNA ligase - Viral capsid proteins Lysozyme and phospholipase which destroy the bacterial cell envelope. Transcription of viral genes occurs in a specific sequence. Immediate-early genes are transcribed first, followed by delayed-early and late genes. RNA polymerase of

102

Animal Virus

4.6.5

the host is used for immediate-early transcription and later is modified chemically by viral enzymes. This modified polymerase is specific for late transcripts. Virion assembly: DNA and capsid proteins self-assemble into virus particles. Lysis: The host cell is destroyed (lysed) after about 25 minutes. About 200 virions are released.

4.6.3. The Lysogenic Cycle of λ phage λ-phage has double-stranded DNA with mutually complementary single-stranded ends (cohesive ends, or “sticky ends”). After viral DNA had entered the host cell, the sticky ends anneal and are joined by the bacterial DNA ligase. At this time the λ phage may proceed along the lytic pathway or follow the lysogenic pathway. In the latter case, it inserts itself into the bacterial chromosome with the help of a virally-encoded integrase enzyme, always between the gal and bio operons. The inserted viral genome is called lysogenic. In the λ-prophage only the gene for the λ-repressor protein is transcribed and translated. The λ-repressor protein prevents the transcription of the other prophage genes. When the cellular level of this repressor protein drops too low, the prophage excises itself from the bacterial chromosome to enter the lytic pathway. This usually happens when the bacterium is exposed to damaging environmental influences such as radiation or heat-stress.

4.6.4. Animal Virus Some typical differences between animal virus and bacteriophages: • Some animal virus replicate in the nucleus and others in the cytoplasm. • After adsorption to the cell surface the complete virus particle enters the cell, often by endocytosis. • Instead of lysing the cell, animal virus are most commonly shed by budding from the plasma membrane, not all virions are released at the same time, and the cell may recover. • Some virus acquire an envelope while budding out of the nucleus or through the plasma membrane. • The immune system can fight viral infections either by forming antibodies to capsid proteins or spike proteins (followed by phagocytosis of the antibody-coated virus), or by destroying the virus-infected cells which display viral proteins on their surface.

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4.6.5. RNA Virus RNA virus can be double-stranded, (+) single-stranded or (-) single-stranded. (+) ss RNA can serve as mRNA while (-) ss RNA is complementary to the mRNA. All RNA virus require a virally-encoded RNA-dependent RNA polymerase (“RNA replicase”). (-) ss RNA virus have to carry at least one copy of this enzyme in the virus particle. Viral RNA replicases have no proofreading ability, therefore the mutation rates are very high.

4.6.6. Retrovirus Retrovirus are enveloped virus containing 2 copies of their (+) -stranded RNA genome in the virus particle together with the viral enzyme reverse transcriptase. Retroviral Life Cycle 1. The nucleocapsid enters the host cell by simple fusion of the viral envelope with the host cell membrane. 2. The reverse transcriptase copies the viral RNA into a double-stranded DNA (a cDNA) which becomes inserted into the host cell genome with the help of a virally-encoded integrase. The inserted viral cDNA contains the viral genes flanked by long terminal repeats which contain the viral promoter. 3. After its insertion into the host cell DNA, the viral DNA directs the synthesis of viral RNA and proteins. The viral RNA is used both for protein synthesis and as the new genomic DNA. 4. New virus particles assemble from viral RNA + protein. These bud out of the cell continuously. Like the RNA replicases, the retroviral reverse transcriptases have a high error rate. Most retrovirus (the AIDS virus is an exception) cannot infect non-dividing cells because they cannot get across the nuclear envelope.

4.6.7. Plasmids Plasmids are semi-independent genetic entities in bacteria, consisting of circular doublestranded DNA carrying a few genes. The genes are not essential for bacterial survival under ordinary conditions but confer special abilities: antibiotic resistance, toxin production, ability to metabolize usual substrates, etc. They also have a replication origin and genes

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regulating their own replication. Most important: R-factors are plasmids that make the bacterium resistant to antibiotics. The plasmid-encoded enzyme β-lactamase (“penicillinase”) destroys penicillin.

4.7. Genetic Recombination 4.8. Types of Recombination The joining of different DNA molecules is called genetic recombination. There are 2 types: Site-specific recombination requires a specific integrase enzyme which recognizes sequences on one or both of the DNA molecules to be joined. The integration of λ-phage and of the retroviral cDNA are examples. General recombination (=homologous recombination) requires no specific sequences, but sequence identity or similarity between the recombining DNAs: transformation in bacteria, crossing-over during meiosis.

4.8.1. Parasexual Processes in Bacteria Bacteria don’t make “real” sex, but they can exchange genetic information through parasexual processes: Transformation is the uptake of foreign DNA. This is random in some species, but other species take up selectively DNA of their own species. The DNA integrates into the chromosome by homologous recombination. Transduction is the transfer of cellular DNA by a bacteriophage. Conjugation is the transfer of DNA by a self-transmissible plasmid. The F-factor of E. coli causes the formation of sex pili which can form a cytoplasmic bridge between the F-factor carrying cell (F+ -cell) and a cell without F-factor (F− -cell). The F factor sends a copy of itself into the F− -cell. Also some R-factors are self-transmissible.

4.9. Objectives in Summary • Describe the covalent structures of DNA and RNA, including their constituent monomers, bond types, functional groups and ionization state. • Describe the structure of the Watson-Crick double helix, with approximate helix parameters, relevant non-covalent interactions, and the rules of base pairing.

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• Define the process of melting of DNA. • Define the different types of nucleases. • Name the different types of DNA polymerases of pro- and eukaryotes and identify their functionally important properties. • Define the semiconservative model of DNA replication. • Identify the proteins involved in prokaryotic DNA replication, and state the sequence of their actions. • Know the functionally important characteristics of prokaryotic and eukaryotic RNA polymerases. • Define the terms “template strand” and “coding strand”. • List the important properties of the genetic code. • Describe the post-transcriptional processing of tRNA, rRNA, and mRNA in prokaryotes and eukaryotes and the general structure of tRNA and the formation of aminoacyltRNA. • Identify the steps in ribosomal protein synthesis, their energy requirements and requirements for soluble protein factors. • Locate the sites of action for some common antibiotics on transcription or translation. • Describe the operon model for the regulation of gene expression in prokaryotes. • Define the principles of positive and negative control of transcription by DNA binding proteins. • Describe the principle events in the life cycles of DNA virus, RNA virus and retrovirus and identify the required viral enzymes. • Describe the properties of histones and their interaction with DNA in nucleosome structure. • Know the approximate proportions of protein-coding, non-coding, unique, moderately repetitive and simple-sequence DNA in the human genome. • Define the terms “promoter” and “enhancer”. • Outline the importance of histones, chromatin structure, DNA methylation and sequencespecific DNA-binding proteins for the regulation of eukaryotic gene expression. • List the types of repetitive elements in the human genome, and describe the mechanism for the mobility of Alu and LINE-1 sequences.

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• Define the terms solitary gene, duplicated gene, gene family, pseudo-gene and processed pseudo-gene. • Describe the importance of telomere erosion and of telomerase for the mortality of somatic cells. • List the mechanisms by which transcription factors can be regulated. • List the principal types of mutations, and predict their likely effects on the functionality of the encoded protein. • Describe the causes of mutations, including replication errors, radiation and the major types of chemical mutagens. • Define the nature and functions of the major DNA repair systems, including the 3’-exonuclease activities of replication complexes, methyl-directed mismatch repair, direct repair, base excision repair and nucleotide excision repair. • Describe the major DNA repair defects, including chromosome breakage syndrome, xeroderma pigmentosum, and Cockayne syndrome. • State the importance of mutations for genetic diseases and carcinogenesis. • Describe the diploid nature of the human genome and its implications for the expression of genetic disorders. • Know the terms “homozygous”, “heterozygous”, “dominant” and “recessive”. • Describe the molecular defect of sickle cell disease, its clinical expression, and the relationship between the two. • Define the terms “α-thalassemia”, “β-thalassemia”, thalassemia major and thalassemia minor. • Describe the clinical presentation in different types of thalassemia. • State the reason for the beneficial effect of increased HbF-expression in patients with sickle cell disease and β-thalassemia, and the beneficial effect of concurrent α-thalassemia minor on the clinical source of sickle cell disease. • List the treatment options for the major hemoglobinopathies.

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5. The Human Genome and Mutations 5.1. The Human Genome The human genome is the complete sequence of DNA bases in the human organism. The finalized drafts of the complete human genome were published in 2001. The human genome was completed after sequencing smaller genomes of model organisms. The entire genome sequences of about 1000 different virus and 100 microbes can be found in the Entrez Genome Browser. The genomes represent both completed genomes and those for which sequencing is still in progress. The three domains of life - bacteria, archaea, and eukaryota - are represented, as well as many virus and organelles. Compared with model organisms the human genome is very large: Organism Genome size (Mb) common name E. coli 4.6 intestinal bacterium S. cerevisiae 12.1 bakers yeast A. thaliana 100 mouse-ear cress D. melanogaster 140 fruit fly M. musculus 3300 mouse H. sapiens 3000 human T. aestivum 17000 wheat The human sequence and its variation is also vital in its importance in medicine. It provides the basis for understanding human disease inheritance and susceptibility. The genome is organized in chromosomes of differing size. Human diploid somatic cells have 22 pairs of autosomes. In addition, females have two X chromosomes, males one X and one Y. Mitochondria also contain DNA which makes up a small fraction of the entire human genome. The unit of DNA which encodes polypeptides is called the gene. In the human genome, there are approximately 30 000 genes. As for most eukaryotes, human genes are not tightly arranged with their nearest neighbors. Very long stretches of DNA occur between genes and also within genes themselves. DNA in humans can be categorized as follows: Unique DNA sequences Protein Coding Regions of Genes (Exons) 1.5 % Non-coding Regions (Introns) 25.0 %

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Other UNIQUE DNA sequences 12.0 % Repetitive DNA sequences All repetitive DNA sequenced to date 53.0 % Not sequenced 8.0 %

5.2. Chromatin Structure 5.2.1. Histones and Nucleosomes Chromatin is about 50 % DNA and 50 % histones. Histones are small, basic proteins (lots of Lys and Arg!) that come in 5 varieties: histones H1, H2A, H2B, H3 and H4. Histones are well-conserved in all eukaryotes. Nucleosomes are formed from a core of histones (2 copies each of histones H2A, H2B, H3 and H4), with about 140 bp of DNA wound around. About 60 bp are in the linker DNA between the nucleosomes. H1 sits on the linker DNA. Higherorder structure: the nucleosomes coil up into a solenoid (diameter 30 nm). In the condensed chromosomes, the 30 nm fibers are attached to scaffold proteins. The DNA in condensed chromosomes consist of DNA:histones:non-histone proteins in 1:1:1 ratio. Euchromatin is dispersed chromatin in which the genes can be transcribed. Heterochromatin is condensed chromatin that cannot be transcribed. The mitochondrial DNA has no histones.

5.2.2. Repetitive DNA There is about 3 × 109 bp of DNA in the human genome, with about 30 000 genes. Only 1.2 % of the DNA codes for proteins. The rest is junk DNA between the genes and in the introns of the genes. Introns are sequences within the genes that are transcribed but not translated. Some of the non-coding DNA is repetitive. Tandemly repeated sequences are most abundant in the centromeric and telomeric regions (“satellite DNA”). Simple-sequence DNA consists of short, tandemly repeated sequences of between 2 and few dozen nucleotides in the repeat unit. Minisatellites and microsatellites are tandem repeats outside the centromeres and telomerase. Interspersed elements are sequences that occur, with variations, in different locations in the genome. Alu sequences are about 300 bp long and are present in about 500 000 copies in the genome (6–8 % of the total genome), with about 80 % sequence homology between different copies. LINE-1 sequences are 6000 bp long. The complete sequence is present in only a few thousand copies, but truncated LINE-1 sequences are very common.

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Genes

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Figure 5.1.: When hybridized with it’s mRNA, the DNA of adenovirus 2 forms loops. These correspond to introns. Image from [Berget et al., 1977].

5.2.3. Mobile DNA Alu sequences and LINE-1 sequences can shuffle their location. These sequences end in an oligo-A tract, and they are framed by short direct repeats. They can jump to new locations by reverse transcription: 1. The element is transcribed into an RNA by RNA polymerase III. This RNA becomes polyadenylated, like an mRNA . 2. Reverse transcriptase makes a cDNA from this RNA. The complete LINE-1 elements contain a gene for a reverse transcriptase. 3. The cDNA is inserted into a new genomic location. These mobile elements are considered selfish DNA. They can cause mutations when they jump into a gene. Another name for mobile element is transposon or less commonly retroposon.

5.2.4. Genes Genes occur mostly in one copy per haploid genome. Duplicated genes are present in 2 or more copies which are close together on the chromosome. Gene families with similar, but nonidentical functional genes are common, usually with closely-related members clustered in the same chromosomal region. Example: globin genes. Pseudo-genes are nonfunctional copies of functional genes. Usually they are close to their functional counterpart. Duplicated genes, gene families and pseudo-genes arise by gene duplication, usually from crossing-over (homologous recombination) between misaligned chromosomes during prophase of meiosis I. Individual exons can duplicate (or be deleted) by the same mechanism.

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Processed pseudo-genes are nonfunctional sequences that duplicate the exon sequences of a functional gene. They are non-viral retroposons that are derived by the reverse transcription of a cellular mRNA (or sometimes a partially processed hnRNA) and the insertion of the resulting cDNA into the genome e.g., reverse transcribed by the Line-1 reverse transcriptase. They are not necessarily close to their functional counterpart. Viral retroposons are the remnants of retroviral genomes that have mutated into non-functionality.

5.2.5. Telomeres Telomeres are the end pieces of the chromosomes. They consist of the repeat sequence TTAGGG, which is repeated hundreds of times to a total length of several thousand bp. They tend to shorten during repeated rounds of DNA replication because lagging strand replication cannot go all the way to the end. Excessive shortening is prevented by the enzyme telomerase, which adds to the repeat sequence by synthesizing new DNA on an internal RNA template. Telomerase is active in the germline and in embryonic tissues, but not in most adult tissues. Therefore the telomeres tend to shorten during a lifetime. This is important for the mortality of cells, both in the body and in cell culture. Cancer cells have telomerase, and they are immortal.

5.2.6. DNA Replication There are many origins of replication in human DNA, about one every 100 000 bp. There are several DNA polymerases: Polymerase α and ζ replicate the lagging and the leading strand, respectively. Polymerase β and  are involved in DNA repair and/or recombination. Polymerase γ is in the mitochondria.

5.3. Mutations Mutations are heritable changes in DNA structure. They arise as errors during DNA replication or are caused by DNA damage. The basal mutation rate is the mutation rate in the absence of external mutagens. Mutations in the germline lead to genetic diseases. Mutations in somatic cells cause cell dysfunction and, sometimes, cancer. All mutagens are also carcinogens.

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5.3.1. Types of Mutation Base substitutions are the most common type. Transition A purine replaces another purine or a pyrimidine replaces another pyrimidine. Transversion A purine replaces a pyrimidine or a pyrimidine replaces a purine. Deletions One or several nucleotides, or a large piece with up to millions of nucleotides, is lost. Insertions One or several nucleotides, or a big chunk of DNA, is added. Translocation A piece of DNA is transferred to another location in the genome.

Other Definitions Point mutations are single-base substitutions. Silent mutations are base substitutions that do not change the amino acid sequence because of the degeneracy of the genetic code. Nonsense mutations change a codon for an amino acid to a stop-codon (UAA, UAG, UGA) resulting in inappropriate termination of chain elongation during translation. Missense mutations change a codon for an amino acid to a codon for a different amino acid. Frameshift mutations are small insertions or deletions that change the reading frame of the mRNA Splice-site mutations result in abnormal splicing. These lead to anything from changes in protein length to frameshifts, which again often lead to truncated proteins.

5.3.2. Causes of Mutations 1. The basal mutation rate is caused by spontaneous tautomeric shifts and hydrolytic reactions. Tautomeric shifts occur with thymine (keto ↔ enol shift) and adenine (amino ↔ imino shift). Spontaneous depurination is the most important type of hydrolytic reaction. 2. UV radiation is a component of sunlight. It causes the formation of pyrimidine dimers and other photoproducts. UV does not penetrate the skin, but it causes sunburn and skin cancer. 3. Ionizing radiation (X-rays, radioactive radiation) is more energy-rich than UV radiation. It causes many kinds of DNA damage, but especially double-strand beaks. Ionizing radiation can penetrate the whole body.

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4. Base analogs are “false” bases that are incorporated into DNA. Example: 5-bromouracil can replace thymine. 5. Deaminating agents deaminate adenine to hypoxanthine and cytosine to uracil. Example: nitrous acid. 6. Deamination of methylated cytosine leads to thymidine, which is not recognized as an unnatural base in DNA, and therefore the repair of T::G mismatches often is erroneous. This is the most common type of point mutation in the genome. 7. Alkylating agents alkylate N and O atoms in the bases. Examples: methyl bromide and ethylene oxide. 8. Intercalating agents are flat, hydrophobic molecules that insert themselves between stacked bases. Examples: acridine dyes and benzene. Ethidium bromide used to visualize DNA in electrophoresis is another intercalating agent. 9. Virus may insert their own DNA into the host cell chromosome, either habitually (retrovirus) or by accident (DNA virus). Mutagens are most effective when they hit the cell immediately before or during DNA replication (during S phase of the cell cycle), because there is no time for repair. Cancer cells are more easily killed by radiation than normal cells because they divide more rapidly and spend more time in S phase.

5.3.3. Mutagenesis Testing The Ames test uses bacteria that are dependent on a particular nutrient as a result of a mutation (auxotrophic bacteria). After exposure to the chemical, the bacteria are screened for back-mutations which restore their ability to grow in the absence of the nutrient. Mutagens increase the rate of back-mutations.

5.4. DNA Repair All cellular organisms have repair systems for many different kinds of DNA damage. DNA repair is possible if only one strand is damaged, therefore the undamaged second strand can supply the sequence information for the repair enzymes. Nucleotide Excision Repair Nucleotide excision repair removes bulky lesions including thymine dimers, alkylations and bulky adducts. The mechanism of excision repair: • A repair crew consisting of several specialized proteins scans the DNA. • The repair complex binds to a bulky lesion.

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• Endonucleases make 2 incisions in the damaged strand, one 5’ and the other 3’ of the lesion. • Helicases in the complex separate the two strands. • The damaged DNA piece is removed. • The resulting gap is filled by DNA polymerase. • The last phosphodiester bond is formed by DNA ligase. • Nucleotide excision repair reaches all parts of the genome, but there is a subsystem for the preferential repair of transcribed genes. Other Repair Systems Depurination (loss of a purine base by spontaneous hydrolysis) is repaired by an AP endonuclease (AP = apurinic), followed by DNA polymerase and DNA ligase. Deaminated bases are removed by base excision repair: hypoxanthine (formed from adenine) and uracil (from cytosine) are clipped off by cleavage of the N-glycosidic bond with D-ribose. The next steps are as in the repair of apurinic sites. Post-replication mismatch repair corrects base mismatches and small insertions and deletions after DNA replication. The repair enzymes can distinguish between the strands because the old strand is methylated on specific sequences (GATC in E. coli, CMG in eukaryotes). The repair system cuts the unmethylated new strand either 5’ or 3’ of the mismatch. The damaged piece is removed by exonucleases and replaced by DNA polymerase followed by DNA ligase.

5.4.1. Repair Defects Xeroderma pigmentosum is a recessively inherited skin disease with premalignant and malignant lesions on sun-exposed skin. There are 9 different types (“complementation groups”), caused by deficiencies of different repair proteins. Cockayne syndrome is also caused by defective nucleotide excision repair, but only the preferential repair of transcribed genes is affected. No skin cancer, but poor growth, neurological problems, and early senility. Hereditary non-polyposis colon cancer (HNPCC) is an inherited cancer susceptibility syndrome caused by defects in post-replication mismatch repair.

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Biochemistry and Genetics

Chromosome breakage syndromes include Bloom syndrome, ataxia-telangiectasia, and Fanconi anemia. These are multi-system disorders with an increased incidence of chromosome breakage.

5.5. Eukaryotic Gene Expression 5.5.1. Transcription There are 3 nuclear RNA polymerases in eukaryotes: RNA polymerase I makes rRNA (in the nucleolus). RNA polymerase II makes mRNA . RNA polymerase III makes small RNAs: tRNA, 5 S rRNA. α-amanitin is a mushroom poison (from Amanita phalloides, the “angel of death”-mushroom) that inhibits RNA polymerase II.

5.5.2. mRNA Processing Eukaryotic mRNA is processed in the nucleus before it is sent into the cytoplasm for translation. Steps: 1. Capping is the addition of a methylguanosine residue to the 5’ end of the mRNA (Figure 8.11 in the Meisenberg book). It occurs co-transcriptionally. The cap protects the 5’ terminus from the action of nucleases. 2. Polyadenylation is the addition of a poly-A tail to the 3’ end of the transcript. Transcription proceeds beyond the polyadenylation signal (consensus: AAUAAA). This is followed by a nuclease cleavage and the enzymatic addition of the adenine nucleotides (no template required!). 3. Splicing is the removal of intron sequences from the primary transcript. It requires spliceosomes which consist of small nuclear ribonucleoproteins (snurps). The intronexon junctions have consensus sequences that are recognized by the spliceosome.

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5.5.3. Translation The important steps are the same as in prokaryotes, but: 1. Eukaryotic proteins start with methionine, not formylmethionine. 2. There is no Shine-Delgarno sequence, but translational initiation depends on interactions of mRNA with ribosomal proteins. 3. Eukaryotic mRNAs are not polycistronic, i.e., they normally encode only one protein. This is normally positioned as the 5’ open reading frame in the mRNA . Eukaryotic translation is inhibited by diphtheria toxin. This toxin modifies an elongation factor covalently, thereby preventing translocation.

5.6. Regulation of Eukaryotic Gene Expression 1. Global Effects: • Histones inhibit transcription non-selectively by tightly binding the DNA doublehelix. Histones are modified by acetylation, phosphorylation and methylation at specific sites. • DNA methylation inhibits transcription. Cytosine bases in the palindrome CG are methylated on both strands, and methylation patterns are maintained during DNA replication. Specific proteins interact with the methylated regions. Not all CG palindromes are methylated: This is regulated. 2. Regulatory DNA Sequences: • Promoter for RNA polymerase II extend to about -200 bp from the transcriptional start site. Highly variable sequence, but most contain a TATA box 25– 30 bp upstream of the start site. Most promoter elements have to be on the correct strand and in the correct 5’ → 3’ orientation. • Enhancers can be present up to some thousand bp upstream or downstream of the transcriptional start site. Enhancers increase the rate of transcription. They need not be in correct 5’ → 3’ orientation to be effective. • Silencers are like enhancers, but they reduce rather than enhance the rate of transcription.

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• Promoters, enhancers and silencers contain binding sites for regulatory proteins. The individual binding sites are often called response elements. Note that in eukaryotes the inhibitory effects of histones and DNA methylation have to be overcome by sequence-specific DNA binding proteins that bind to promoter and enhancer sequences. 3. Transcription Factors • The regulatory proteins that bind to promoters and enhancers are called transcription factors. General transcription factors are promoter-binding proteins that are required for the transcription of all genes by a particular RNA polymerase. They are the functional equivalents of the bacterial σ-subunit. Others affect the transcription of some genes but not others. • Many transcriptional regulators bind DNA in a dimeric form, either as homodimers or as heterodimers of 2 slightly different polypeptides. Recognition occurs in the major groove where the ’side-on’ steric features of base-sequences can be recognized by proteins. Types: Zinc-finger proteins (see fig 4.1 on page 93) contain between 2 and about a dozen zinc fingers in their DNA-binding region: zinc complexed between 4 Cys or 2 Cys and 2 His residues, with an intervening loop. Leucine-zipper proteins contain a DNA-binding basic domain, a dimerization domain with leucine residues spaced 7 amino acids apart in an amphipatic α-helix, and a transcriptional activator (or repressor) domain. Helix-loop-helix proteins have a dimerization domain with 2 amphipatic αhelices separated by a loop. Helix-turn-helix proteins have 2 α-helices separated by a β-turn. One of the α-helices fits into the major groove of the DNA. • Regulation of Transcription Factors a) The synthesis of transcription factors is controlled often in a cell type-specific manner: most transcription factors (except the “general” transcription factors) are present only in certain cell types and at certain stages of cell differentiation. b) They can be controlled by the reversible binding of small molecules. Example: receptors for steroid and thyroid hormones. c) They are controlled by interactions with other proteins. d) They are controlled by phosphorylation and dephosphorylation, frequently in response to growth factors or the second messengers of hormones.

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5.7

4. Post-transcriptional Controls a) The use of alternative promoters or alternative polyadenylation signals can create mRNAs which differ at the 5’ end or the 3’ end, respectively. b) Alternative splicing of a single mRNA transcript can produce different polypeptides from the same gene. c) RNA editing is the post-transcriptional chemical modification of a base in the mRNA . The example, an amino acid coding codon can be modified into a stop codon to produce a shorter protein. d) mRNA stability varies in different cell types and under different conditions. mRNA stability can be controlled by specific mRNA sequence binding-proteins that impair the action of nucleases. 5. Translational Controls a) Start-site variation occurs when alternative start codons can be used by the ribosome. The resulting polypeptides differ at their N-terminus. b) Initiation factor control works through the phosphorylation of the initiation factor eIF-2 which slows all translation. This occurs during the M phase of the cell cycle, and when growth factors are not present. c) Cap-binding proteins can inhibit translation by binding to the 5’ end of the mRNA .

5.7. Objectives in Summary 1. Know the proportion and characteristics of DNA sequence elements in the human genome. 2. Describe the properties of histones and their interaction with DNA in nucleosome structure. 3. Define the terms promoter, enhancer, and silencer. 4. Explain how histones, chromatin, DNA methylation, and transcription factors can influence the rate of eukaryotic transcription. 5. Name and describe the principal types of mutations, and explain their effects on the structure and function of proteins. 6. Describe the causes of mutations. 7. Name and describe the systems for repair of DNA lesions.

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8. List inherited human diseases which result from faulty DNA repair systems. 9. Name the different types of eukaryotic DNA polymerases, and describe replication. 10. Know the different types of eukaryotic RNA polymerases, and accessory factors. 11. Describe the processes of post-transcriptional processing of tRNA, rRNA and mRNA in eukaryotes.

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6. Chromosome Aberrations 6.1. The Human Karyotype Chromosomes become visible only during mitosis and meiosis. They are classified as: Metacentric the centromere is in the middle. Submetacentric intermediate between metacentric and acrocentric. Acrocentric centromere near the end, forming a stubby short arm and a long arm. Only the long arm has genes. Telocentric the centromere is at the end; only one arm is present. Does not occur in humans. The short arm of a chromosome is designated by the letter p (petite, French for small, the long arm by q. The acrocentric chromosomes have small satellites attached to their short arms, which contain clusters of genes for ribosomal RNA; losing one or two of these clusters will not affect the cell in any adverse way. Diploid somatic cells have 22 pairs of autosomes. In addition, females have two X chromosomes, males one X and one Y. The karyotype (= chromosome constitution) can be examined during mitotic metaphase. Techniques: • Bone marrow biopsy (no culture required) • Leukocyte culture • Fibroblast culture from the skin • Amniotic cells, cultured like skin fibroblasts Leukocyte culture is most important. Phytohemagglutinin (a lectin from beans) is used as a mitogen, and colchicine is used to arrest mitosis in metaphase. Human chromosomes can be grouped into: Group A (1-3) large, metacentric to submetacentric. Group B (4-5) submetacentric, smaller than group A. Group C (6-12) submetacentric, smaller than group B.

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Group D (13-15) acrocentric Group E (19-18) submetacentric Group F (19-20) small metacentric Group G (21-22) small acrocentric X chromosome like a C-group chromosome Y chromosome small submetacentric Banding techniques are staining procedures that distinguish between different types of chromatin within the chromosomes: Q (quinacrine mustard) banding, G (Giemsa) banding, and R (reverse) banding. They can distinguish different chromosomes within each group, and they can detect structural abnormalities. Karyotype with banding costs about twice as much as a regular karyotype. High-resolution banding with prophase chromosomes is not often done in clinical routine, but is an important research technique.

Chromosome painting makes use of fluorescent-labeled probes that bind to one of the chromosomes (as in FISH = fluorescent in-situ hybridization; FISH is discussed in the section on recombinant DNA methods). Different-colored probes are used to distinguish between chromosomes. Fluorescent probes can even be used to detect aneuploidy in the interphase nucleus. This method tends to supplement or even replace classical karyotyping. Heteromorphisms are variations in the karyotype that are in most cases not associated with disease: • Length variations of the long arm of the Y chromosome (Yq) are present in 10 % of all males. Yq is mostly junk DNA, and most of these variants are innocuous. • The satellites of the acrocentric chromosomes are variable. Also these heteromorphisms are generally asymptomatic. • There are fragile sites on many chromosomes, which are visible as small constrictions. Chromosome breakage at these sites can be induced by culturing in a folate-deficient medium. Many fragile sites are variable, and most (but not all) of them are harmless. • Centromeric heterochromatin, and also heterochromatic bands outside the centromeres, can be variable. Question: What should you do when you pick up a heteromorphism incidentally during prenatal diagnosis?

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6.2. Sex Chromatin In interphase nuclei, one of the two X chromosomes of a female is frequently visible as a heterochromatic mass, the Barr body. Number of Barr bodies in diploid somatic cells = number of X chromosomes minus 1: Normal females have one, normal males none. Sex chromatin in buccal smear cells is an important screening test for sex chromosome aberrations. The Lyons hypothesis states that only one of the X chromosomes in a cell is in the dispersed, genetically active form during interphase. Additional X chromosomes become heterochromatic and genetically inactive. This involves DNA methylation. Inactivation occurs about 15–16 days post-conception and is random. Inactivation can affect either the maternallyderived or the paternally- derived X chromosome: normal females are mosaics of cell clones in which either one or the other X chromosome is active. This is important for the expression of X-linked diseases in heterozygous females. The genes in the pseudo-autosomal region on the short arm of the second X chromosome escape inactivation, and also some other genes remain active. Therefore people with an abnormal number of X chromosomes do have clinical abnormalities. Only the few genes in the pseudo-autosomal region have an equivalent on the Y chromosome. The chromatin of the Y chromosome can be demonstrated in the interphase nucleus by Q-banding. This method can be used to distinguish X- and Y-bearing sperm cells. Most of the long arm of the Y chromosome is heterochromatic and non-coding. The short arm carries the male-determining SRY gene, which directs the development of the testis. It codes for a transcription factor. There are also a few Y-linked genes that are required for spermatogenesis, and mutations in these genes can lead to male infertility.

6.3. Types Of Chromosome Aberrations Gross chromosomal aberrations are present in 1 out of every 160 live births and 35 % of spontaneous abortions. More than half of all pre-implantation embryos are mosaics with at least one abnormal cell line. These chromosome aberrations are a major reason why even for young women the risk of pregnancy is never higher than 25 % to 39 % per month. About 25 % of women with primary amenorrhea, 11 % of infertile males and 10 % of the institutionalized mentally impaired have chromosomal abnormalities. Numerical aberrations: Aneuploidy: One chromosome is present in an abnormal copy number: trisomy, monosomy. Polyploidy: The whole set is present in an abnormal number: triploidy (69 chromosomes total), tetraploidy (92 chromosomes).

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6.4.1

Biochemistry and Genetics

Consequences: • Polyploidy results in fetal death. • Monosomy for an autosome results in a nonviable embryo, trisomy is better tolerated. • Trisomy of large autosomes causes death, only trisomies of some of the smaller autosomes are viable. • Aneuploidy of sex chromosomes is well tolerated, but at least one X chromosome has to be present. • People with a Y chromosome are usually male, those without a Y chromosome are usually female. Aneuploidy results from non-disjunction during meiosis, or during mitosis in the germ line. If it happened in a mitotic division of spermatogonia or oogonia, there is germline mosaicism: presence of a whole tribe of abnormal cells in the gonad. This means there is an increased recurrence risk after the birth of an affected child. Aberrations of chromosome number generally are not inherited from an affected parent, but arise de novo. Mosaicism is caused by non-disjunction during the first mitotic divisions after fertilization: different somatic cells of the patient have different karyotypes. Chromosomal rearrangements are aberrations of chromosome structure, such as large deletions or translocations. They often arise de novo, but can also be inherited.

6.4. Autosomal Trisomies Only 3 autosomal trisomies are observed in live-births: Trisomy 21: Down syndrome About 1 in 800 births. Trisomy 18: Edward syndrome About 1 in 8000 births Trisomy 13: Patau syndrome. About 1 in 20 000 births.

6.4.1. Trisomy 21 (Down syndrome) 95 % of Down syndrome patients have trisomy 21, 3 % a Robertsonian translocation (“translocation-type Down syndrome”), and 2 % are mosaics with trisomic and normal cells. In 80 % of the cases non-disjunction occurred in the mother, usually in meiosis I. About 60 % of trisomy 21 conceptuses abort spontaneously. Phenotype: • Hypotonia (infants)

124

Trisomy 21 (Down syndrome)

6.4.2

• Short stature • Short, stubby hands and feet • Palpebral fissure, epicanthic fold • Large, protruding tongue • Small or malformed ears • Flat occiput • Short, broad neck • Simian crease • IQ typically between 25 and 50 • Infertility in males There is an increased risk of: Congenital heart defects (30–40 %): Endocardial cushion defect, atrial and ventricular septal defects. Childhood leukemia is 10–20 times more common than in euploid individuals. Both myelogenous leukemia (infants) and lymphoblastic leukemia (older children) are increased. Dementia in older patients. Life expectancy is reduced, but many patients live into their 50s and 60s. The neuropathologic changes of Alzheimer’s disease (plaques and tangles) are present in patients dying at > 35 years. The β-amyloid precursor protein (APP) gene encoding one of the components of Alzheimer plaques is located on chromosome 21. Maternal Age frequency Very young mother 1 in 1500 Mother 35 years 1 in 365 Mother 40 years 1 in 100 Mother 45 years 1 in 25 Paternal Age has only a very slight effect (if any, the effect is delayed 20 a relative to the maternal age effect). Prenatal diagnosis: Karyotype analysis after chorionic villus sampling or amniocentesis. Also maternal serum α-fetoprotein (reduced in Down syndrome). The recurrence risk after the birth of a trisomy 21 child to a young mother is about 1 % because of possible germline mosaicism.

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6.5.1

Biochemistry and Genetics

6.4.2. Trisomy 18 (Edward syndrome) • 80 % of patients are female • Birth-weight under 6 pounds • Failure to thrive • Hypertonicity, flexed fingers • Prominent occiput • Low-set, malformed ears • Micrognathia • Short sternum, small pelvis, rocker-bottom feet • Cardiac and renal malformations • 90 % die within a year • Profound mental deficiency in survivors

6.4.3. Trisomy 13 (Patau syndrome) • Apparent deafness • Minor motor seizures • Hypertonia/flexed fingers • Sloping forehead, microcephaly • Eye abnormalities • Cleft lip, cleft palate • Agenesis of the olfactory bulb, arhinencephaly, holoprosencephaly • Cardiac/colon/uterine malformations • Polydactyly • Rocker-bottom feet • 90 % die within a year • Profound mental deficiency in survivors

126

XX Males

6.6.1

6.5. Sex Chromosome Aberrations: Male 6.5.1. Klinefelter syndrome Incidence 1 in 1000 males, mild maternal age effect. Chromosomes 80 % are 47, XXY. The others are mosaics (46, XY/XXY most common), 48, XXXY, or 49, XXXXY. Phenotype (47, XXY): Small testes are the most consistent physical finding. Infertility. Height is average or above average. Undervirilization, with poor growth of beard and body hair, penis small or normal, 25 % develop gynecomastia. Testosterone is low, estrogen variable, pituitary gonadotropins high. IQ is decreased by about 15 points. More severe abnormalities (with mental deficiency) in 48,XXXY and 49, XXXXY. Diagnosis Most patients present either with poor sexual development or infertility. Buccal smear and/or karyotype is indicated in these situations. Klinefelter is the single most important cause of severe male infertility. Treatment: Testosterone improves virility but not fertility.

6.5.2. XYY constitution (“murderer chromosome”) Incidence 1 in 1000 males. No parental age effect. Phenotype No consistent abnormalities, except: • Increased height • IQ reduced by 10–15 points • Increased risk of criminal conviction XYY males are over represented in prisons (3 in 1000), institutions for the mentally defective (3 in 1000) and institutions for mentally defective criminals (20 in 100), but most are never diagnosed. Normal fertility; children are normal. Presence of the additional chromosome does not constitute a legal defense.

6.5.3. XX Males Rare (1 in 20 000). Usually caused by a small translocation of the testis-determining gene to the X chromosome. Klinefelter-like phenotype.

127

6.7.1

Biochemistry and Genetics

6.6. Sex Chromosome Aberrations: Female 6.6.1. Turner’s syndrome (gonadal dysgenesis) Incidence 1 in 5000 females Chromosomes 50–60 % are 45, XO. Others are mosaics (46, XX/45, XO; 47, XXX/45, XO) or structural rearrangements. 99 % of XO fetuses abort spontaneously. Gonads “Streak ovaries”, consisting of connective tissue without oogenesis. Degeneration of the ovaries starts during fetal development. At birth Peripheral lymphedema (first few days only, webbing of neck, 20 % have aortic coarctation. Later Short stature, final height is 140–1 50cm. Broad chest, widely-spaced nipples, cubitus valgus, low posterior hairline. At puberty There is no puberty. Mental development is about normal. Diagnosis Buccal smear, karyotype. Some are diagnosed before puberty because of slow growth, others are diagnosed later during workup of primary amenorrhea. Treatment: Estrogen after puberty, growth hormone in younger patients.

6.6.2. Triple-X (47, XXX, “superfemale”) Incidence 1 in 1000 females, maternal age effect. Phenotype No consistent abnormalities, but IQ is decreased 10–15 points, and some have impaired fertility or menstrual problems. Their children are usually normal. 48, XXXXX are rare, with mental deficiency and dysmorphic features.

6.6.3. XY females Rare (1 in 20 000), caused by deletion of the testis-determining gene. Turner-like phenotype.

128

Female Pseudohermaphroditism.

6.7.3

6.7. Abnormal Sexual Development With Normal Chromosomes 6.7.1. True hermaphroditism Definition Presence of ovarian and testicular tissue, either as separate gonads or combined in an ovotestis. Incidence Very rare. Phenotype A variable mix of male and female structures. Chromosomes Most are 46, XX. Some are mosaics with a Y-containing cell line, some are chimeras (created by the fusion of two embryos).

6.7.2. Mixed Gonadal Dysgenesis Definition Presence of testis and streak ovary. Incidence Rare Phenotype Variable. Often virilization at puberty. Chromosomes Most are mosaics 46, XY/45, XO.

6.7.3. Female Pseudohermaphroditism. Definition Virilized phenotype in a female. Only ovaries are present. Causes Prenatal androgen or progesterone treatment. Or a recessively inherited deficiency of 11 — or 21 — hydroxylase in the adrenal cortex: congenital adrenal hyperplasia (“adrenogenital syndrome”, incidence 1 in 10 000). The synthesis of corticosteroids is blocked while androgen synthesis is possible.

129

6.7.5

Biochemistry and Genetics

Pituitary ACTH

+ Cholesterol

Progestins

Corticosteroids

Androgens

The lack of corticosteroids causes excessive ACTH secretion and stimulation of the adrenal cortex overproducing progestins and adrenal hyperplasia. With corticosteroids synthesis blocked, the progestins are converted to androgens. These adrenal androgens make virilization in females and precocious puberty in males. If the enzyme deficiency is complete, there is dangerous hyponatremia and hyperkalemia because mineralocorticoids are missing. Cortisol treatment permits normal development both in males and females. Early diagnosis is important.

6.7.4. Male Pseudohermaphroditism Definition Feminized phenotype in a genetic male. Causes Defective androgen action. Androgen insensitivity (testicular feminization) is a rare X-linked recessive disorder. The external phenotype is female. Female body proportions, breast development, feminine psychosexual development. No uterus and fallopian tubes, primary amenorrhea, no Wolffian duct structures, non-functional testes are present in abdomen. Sometimes an “inguinal hernia” turns out to be a testis. Otherwise after puberty when amenorrhea or infertility is the presenting problem. The testes are prone to malignancies and should be removed. There is a whole spectrum of incomplete androgen insensitivity, ranging from infertile male to phenotype female. 5α-reductase deficiency a rare autosomal recessive condition, prevents the formation of dihydrotestosterone from testosterone. Dihydrotestosterone is the most potent androgen. The external genitalia of a genetic male are mostly female at birth but internal Wolffian duct structures are present. Virilization occurs at puberty.

130

Chromosomal Rearrangements

6.7.5

6.7.5. Chromosomal Rearrangements Chromosomal rearrangements are caused by chromosome breakage, often followed by faulty healing of the fragments. To be stable during mitosis, the rearranged chromosomes must have one centromere and the telomeres. Balanced rearrangements do not change the copy number of genes, therefore their phenotype is normal. Genes can still be expressed, even on the wrong chromosome, as long as they still have their promoter and enhancers. Unbalanced rearrangements lead to a net excess or deficiency of genes, therefore the phenotype is abnormal.

Deletions Both terminal and interstitial deletions occur, and they come in all sizes.

Turner syndrome Deletions in the short arm of the X chromosome (Xp-) make typical Turner syndrome. Deletions of the long arm (Xq-) make only streak gonads and primary amenorrhea. Autosomal deletion syndromes Usually with a combination of physical abnormalities and mental deficiency. Larger deletions (> 10 000 000 bp) can be seen in the banded karyotype, and smaller deletions can be diagnosed with FISH. Examples: Cri-du-chat syndrome is caused by deletion of part of the short arm of chromosome 5 (5p-). Physical stigmata are mild, but there is severe to profound mental retardation. Infants have a cat-like cry. 80 % of patients are female.

131

6.7.5

Biochemistry and Genetics

Williams syndrome is caused by an interstitial deletion in the long arm of chromosome 7 (7q-). Patients are pixie-faced, with short stature, transient hypercalcemia, and mental retardation but spared verbal ability.

Ring chromosomes

This is a rare type of abnormal chromosome in which end pieces of the chromosome are lost while the ring is formed:

Isochromosomes

Isochromosomes consist of two identical chromosome arms. They are formed by transverse rather than lengthwise cleavage of the centromere during the metaphase to anaphase transition in meiosis II or mitosis.

IsoXq causes Turner’s syndrome. Isochromosomes of autosomes (other than acrocentrics) are not compatible with life.

132

Chromosomal Rearrangements

6.7.5

Inversions

Inversions are either pericentric or paracentric. They are usually asymptomatic, but unbalanced offspring can be produced if crossing-over occurs in the inverted segment during meiosis.

Reciprocal Translocations

Breaks occur in two non-homologous chromosomes. The fragments unite to form two abnormal chromosomes:

133

6.7.5

Biochemistry and Genetics

Carriers of a reciprocal translocation are normal, but gametes with abnormal chromosomes may be produced, resulting in unbalanced translocations in the offspring: High incidence of stillbirths and spontaneous abortions. Robertsonian Translocation (=centric fusion) This is a fusion of the long arms of two acrocentric chromosomes after breakage near the centromere.

The small fragment is lost. This consists of genetically inactive heterochromatin, as well as rRNA genes. Normally, other rRNA loci are present and functional and therefore no phenotype is expected.

134

Objectives in Summary

6.8

The carriers of a Robertsonian translocation have only 45 chromosomes. They are phenotypically normal, but can produce abnormal offspring. Example: Translocation-type Down syndrome is present in 3 % of unselected Down syndrome patients. Typical features: • Clinical severity is the same as trisomy 21 • There is no maternal age effect • The cases are often familial • The propositus has 46 chromosomes • In many cases one of the parents (usually the mother) has 45 chromosomes. Offspring derived from a gamete carrying both the translocation chromosome and a normal chromosome 21 (but not the normal copy of the chromosome to which chromosome 21 is attached) will have translocation Down syndrome. In an asymptomatic carrier of a Robertsonian t(21;21) translocation (isochromosome 21), all children will have Down syndrome.

6.8. Objectives in Summary 1. Define polyploid, aneuploid, monosomy, trisomy, deletion, translocation, balanced rearrangement, unbalanced rearrangement, ring chromosome and isochromosome. 2. Recall the approximate incidence values of chromosome aberrations. 3. Describe the causes of aneuploidy, mosaicism and chromosomal rearrangements. 4. Describe the normal function of the X and Y chromosomes in sex determination. 5. Provide examples of clinical situations in which sex chromatin determination should be performed. 6. Describe methods for karyotyping and give examples of when a karyotype should be performed. 7. List the phenotypic features associated with major chromosome aberrations and also the more common trisomies and sex-chromosome ploidies. 8. Predict Down syndrome incidence based on maternal age. 9. Given a patient with a reciprocal (balanced) translocation, state the likely consequences for the translocation carrier and future children.

135

6.8

Biochemistry and Genetics

10. Describe the clinical definitions of hermaphroditism, male/female pseudohermaphroditism, mixed gonadal disgenesis. 11. Describe the pathogenesis in testicular feminization and adrenogenital syndrome. 12. Identify clinical signs and symptoms suggesting unbalanced chromosomal rearrangements.

136

7. Enzymes Life is an ordered sequence of enzymatic reactions (R. Willstätter )

7.1. History of enzymology Biotechnological use of enzymes in food production is a very ancient part of human culture. The Codex Hamurabi (≈ 2100 BC) mentions the production of wine by fermentation, but pottery used to drink wine or beer from has been found from much earlier dates. Homer (≈ 600 BC) in the 5th song of his Iliad mentions the use of ficin (fig tree extract) in cheese production, the first written record of the use of (semi-)purified enzymes. Scientific work on enzymes started at the end of the 18th century with Reaumur and Spalanzani, who noted that the stomach juice of buzzards and seagulls would digest meat and soften bone, but would not act on plant material (enzymes are substrate specific) and that the activity was lost on storage (enzymes are unstable). The first to note the macromolecular character of proteins was J.F. Engelhart, who in 1825 found the iron content of hemoglobin to be 0.334 % of the total mass, irrespective of species. Since iron has an atomic mass 55.8 Da, the molecular mass of hemoglobin must be n × 16.7 kDa, with n the number of iron atoms in a hemoglobin molecule (now known to be 4). This “hasty conclusion” drew a lot of ridicule from his contemporaries, who refused to believe that any molecules could be that big. Nevertheless G.J. Mulder in 1838 coined the term protein, which is derived from Greek and means “of prime importance”. M. Traube suggested in 1877 that enzymes are proteins, but the idea that these “colloids” — with which to work was below the dignity of any self-respecting chemist — could have such a fundamental role was not accepted until J.B. Sumner crystallized pure urease in 1926. Today we know that although most enzymes are indeed proteins, some RNAs also have catalytic properties (“ribozymes”), a discovery made by T. Cech in 1980. By the end of the 19th century it had become clear that both animals and plant produce powerful enzymes, which allowed degradation of biological material in the test tube under milder conditions (pH, temperature) than those required by a chemist. It was however believed that the production of complex living matter could be performed only by the organized ferments within a cell, which needed a special “vis vitalis” (Lat.: force of

137

7.2.2

Biochemistry and Genetics

life) to perform such reactions. This assumption is called vitalism. In 1897 Hans and Eduard Buchner tried to preserve a cell-free juice pressed from yeast by addition of household sugar, so they could market it for health purposes. Addition of sugar or salt was a common preservation method before refrigerators became available. To their surprise however the yeast juice was able to ferment the sugar under production of carbon dioxide, a reaction that vitalists had claimed would be possible only in a living cell. Their chance discovery opened the way for reductionism, the assumption that living organisms are governed by the same laws of physics and chemistry that hold in a test tube. Reductionism distinguishes modern medicine from shamanism and simple quackery. With this discovery the distinction between ferments and enzymes lost its meaning, today we use both terms synonymously.

7.2. Classification of enzymes 7.2.1. Systematic name In the 19th century naming an enzyme was the privilege of its discoverer. This resulted in names that had little systematic meaning, some of them we even use today as trivial names: trypsin, diastase and so on. However, given that the average cell contains some 2000 different enzymes this way of naming enzymes became rapidly untenable. Today we name enzymes by taking the names of their substrate, adding the name of the reaction performed and the ending “ase”. For example acetylcholine esterase is an enzyme that cleaves the ester bond in the neurotransmitter acetylcholine, producing acetic acid and choline. Water as substrate is not included in the name, thus it is acetylcholine esterase rather than acetylcholine:water esterase.

7.2.2. Enzyme classes and EC codes It turns out that enzymes perform only 6 classes of reactions: 1) Oxidoreductases catalyze the transfer of electrons, hydrid ions or hydrogen atoms between molecules: H3C C OH H2 ethanol

138

+

NAD

+

NAD+: alcohol

oxidoreductase

NADH

+

H

+

+

H3C C O H ethanal (acetaldehyde)

7.2.2

Enzyme classes and EC codes

2) Transferases catalyze the transfer of functional groups between molecules 2-

H2C OH

H2C O PO3 O

OH

+

OH OH

O

ATP : glucose

ATP

phosphotransferase

+

ADP

OH

OH

D-glucose

OH

OH

Glucose-6-phosphate

OH

3) Hydrolases catalyze the transfer of functional groups to water 2-

H2C O PO3 O

H2C OH OH

+

OH OH

O

glucose-6-phosphatase

H2O

OH

+

OH

2-

HPO4

OH OH

glucose-6-phosphate

D-glucose

OH

4) Lyases form double bonds by removing functional groups from a molecule HC COO

C COO C COO H

-

-

-

+

aconitate

H2 O

hydratase

HC COO

HC COO HO C COO H

-

-

-

iso-citrate

cis-aconitate

5) Isomerases transfer functional groups within a molecule HC

O

H2C

HC OH

glucose-6-phosphate

HO CH

isomerase

HC OH HC OH

OH

C O HO CH HC OH HC OH

2-

2-

C O PO3 H2

C O PO3 H2

6) Ligases use the chemical energy of ATP-hydrolysis to form C-C, C-O, C-N or C-S bonds COO C O

+

COO OH

-

+

CH3

Pyruvate

ATP

pyruvate carboxylase

COO C O

+

ADP

+

Pi

CH2

COO -

Bicarbonate

Oxaloacetate

The Enzyme commission, responsible for naming of enzymes within the IUBMB/IUPAC has introduced a 4-figure code number to uniquely identify each enzyme. Example: ATP:glucose phosphotransferase 2.7.1.1 (glucokinase): the enzyme is a transferase (class 2), a phosphotransferase (subclass 7), transfer is to a hydroxy-group (1). The fourth number has no systematic meaning, it simply allows the unique identification of each enzyme.

139

7.3.1

Biochemistry and Genetics

Chemical and biological direction of a reaction We have learned in the thermodynamics section of this course that all reaction are, at least in principle, reversible, and that catalysts only accelerate the establishment of the equilibrium, without influencing the equilibrium constant. Chemists by convention write a reaction so that when it proceeds from left to right ∆G0 is negative, for example: ATP + H2 O * ∆G00 = −30.5 kJ/mol. ) ADP + Pi Within the mitochondria of a cell however the reaction runs in reverse direction, using the energy liberated by oxidation of food to drive it. Thus the biological direction of the reaction is (in this case) opposite to the chemical. Nevertheless, enzymes are named by chemical direction, hence the enzyme is called systematically ATPase rather than ATP synthase.

7.3. Kinetics The first to develop a mathematical relationship between substrate concentration and reaction velocity of an enzymatic reaction was the French physicochemist V. Henri in 1902. Because the influence of the hydrogen ion concentration on enzyme activity was not known at his time however, measurements did not follow the predicted values well. Once P.L. Sørensen defined this role, introduced the pH-scale and the concept of buffering, L. Michaelis and his postdoc M.L. Menten revisited the field and confirmed Henri’s result in 1913. In 1925 G.E. Briggs and J.B.S. Haldane came to the same result in a more rigorous way.

7.3.1. The Henri-Michaelis-Menten (HMM)-equation To derive this relationship, we start with the assumption of E. Fischer (1894) that enzymes act by binding their substrate in a special pocket, called binding site, into which the substrate fitted like a key into a lock: k+1 k+2 k+3 GGGGGGGB GGGGGGGB GGGGGGGB E + SF GG ES F GG EP F GG E + P k−1 k−2 k−3 Within the enzyme the substrate is turned over into product and finally released. If we perform the reaction in the absence of product, then the release step becomes irreversible, giving us: k+3 k+1 k+2 GGGGGGGB G G G G G G G B E + SF ES EP G G G GGGGAE + P GG F GG k−1 k−2 The conversion of S into P inside the enzyme is not accompanied by any binding or release steps, it is therefore independent of the concentrations of the reactants and can not be

140

7.3.1

The Henri-Michaelis-Menten (HMM)-equation

Figure 7.1.: Phases of an enzymatic reaction. in the first phase (pre-steady state) the rate of product formation is low because the ES-complex must be formed. In the second, steady-state phase the rate of ES formation from E + S is equal to the rate of its breakdown into E + P, [ES] is constant and [P] increases linearly over time. In the last phase the substrate concentration becomes so low that ES-formation slows down, leading in turn to a decrease in the rate of product formation. Note the logarithmic time scale. Figure taken from [Buxbaum, 2007]. time-course of an enzymatic reaction 1

concentration (rel. units)

0.8 [S] [E] [ES] [P]

0.6

0.4

0.2

0 1

10

non-steady state

100 time (rel. units)

steady state

1000

10000

depletion phase

investigated by kinetic methods. Hence one can further simplify the reaction scheme: kcat k+1 GGGGGGGB E + SF GG ESGGGGGGGAE + P k−1 If we mix enzyme and substrate, the ES-complex will begin to form. Initially, the rate of formation of P will be zero, because there is no ES (law of mass action, see section 2.2 on page 19). As [ES] increases, the rate of its breakdown into E + P will also increase, until an equilibrium is established, where the rate of formation of ES from E + S is equal to its rate of breakdown into E + P (see fig. 7.1). The Henri-Michaelis-Menten (HMM)-law of enzyme kinetics, which we are about to derive, applies only in the steady-state phase of the reaction. The rate at which product is formed is, according to the law of mass action, given by v = kcat × [ES]

(7.1)

141

7.3.1

Biochemistry and Genetics

Figure 7.2.: Left: Plot of the Henri-Michaelis-Menten (HMM)-equation. Right: Relationship between the HMM-equation and a hyperbola. Figures taken from [Buxbaum, 2007]. normalised Henri-Michaelis-Menten curve

1

v

y

y

v / Vmax

0.75

2

y2 = x - a2

0.5

S

x

0.25

x 0 0

1

2

3

4

5 [S] / Km

6

7

8

9

10

In the absence of the second reaction step, [ES] would depend on [E], [S] and Kd = k−1 /k+1 = 1/Keq : [E] × [S] [ES] = (7.2) Kd where Kd is the concentration of substrate where [ES] is half the total enzyme concentration [E]t , in other words [E] = [ES]. Since however in an enzymatic reaction ES can break down not only into E + S, but also into E + P, [ES] must be lower than predicted by equation 7.2. G.E. Briggs and J.B.S. Haldane have shown in 1925 that equation 7.2 can still be used, however, that we have to replace the dissociation constant Kd = k−1 /k+1 with the Michaelis-constant Km = k−1 +kcat . While Kd is the concentration of substrate where in the absence of turn-over half k+1 the enzyme molecules have substrate bound, Km is the substrate concentration where the rate of the enzymatic reaction is half maximal. If we replace [ES] in eqn. 7.1 with the modified equation 7.2 and rearrange, we get the HMM-equation: v=

kcat × [E]t × [S] V × [S] = max Km + [S] Km + [S]

(7.3)

This equation is graphed in fig. 7.2. We note that • if [S] = ∞, all enzyme will be present as ES ([ES] = [E]t ), and the rate of product formation will approach the maximum kcat × Et . • if [S]  Km , relative large changes in [S] will have only a small effect on v, in other words, the reaction will be of zeroth order with respect to S. Example: If [S] = 10×Km , 10Kd = Vmax × 0.91. If on the other hand [S] = 20 × Kd , then then v = Vmax × 11K d

142

The Henri-Michaelis-Menten (HMM)-equation

7.3.1

v = Vmax × 0.95. In other words, a doubling of the substrate concentration resulted in a 4.4 % increase in v. • if the [S] = Km , then v =

Vmax ×Km 2Km

= Vmax /2

• if [S]  Km , then the relationship between [S] and v can be approximated by a straight line v ≈ [E] × [S]. The unit of the velocity of an enzyme reaction is the katal, 1 kat = 1 mol/s. In the older literature you may still find the enzyme unit, 1 U = 1 µmol/s = 16.7 nkat. Application: Forensic determination of blood alcohol concentration In a considerable percentage of automobile accidents one or several of the drivers involved are under the influence of intoxicating spirits. Because alcohol has a very detrimental effect on the ability of motorists to control their vehicle, most countries have legal limits on [alcohol] in the blood of motorists. Depending on jurisdiction, these tend to vary between 0.2–0.8 h, but some countries enforce a strict 0 h rule. In an “untrained” drinker 1.5 h leads to unconsciousness, 4 h to death by respiratory suppression.

Ethanol is oxidized in our livers by alcohol dehydrogenase, which has a Km of 1 mM ≡ 0.046 h. In other words, at legally relevant blood alcohol concentrations the enzyme works near substrate saturation, the reaction is of zeroth order with about 0.15–0.2 h/h. There is usually a considerable delay between an arrest for drunk driving and the taking of a blood sample, but this relationship allows the back-calculation of the alcohol concentration at the time of arrest or the time of an accident (which is the legally relevant one). The Lineweaver-Burk-transformation If you have experimentally determined the reaction rate v of an enzyme as function of the substrate concentration [S], it is not always easy to determine Vmax , because the curve at high substrate concentrations is so flat. This is especially true if your data points contain experimental error (which they always do!). Woolf had the idea to linearize the HMM-equation by using either one of three mathematical transformations, but his work was largely ignored. Later these transformation were re-discovered by other workers, whose name they now bear. The most important one is the Lineweaver-Burk-transformation, which consists of taking reciprocals: v=

Vmax × [S] Km + [S]

⇒

1 1 K 1 = + m × v Vmax Vmax [S]

(7.4)

This is the equation of a straight line (y = a + b × x), the y-intercept is 1/Vmax , the slope is Km /Vmax and, as can be shown easily by setting to 0, the x-intercept is −1/Km .

143

7.3.2

Biochemistry and Genetics

Figure 7.3.: Lineweaver-Burk-transformation of v vs. [S] data results in a straight line, but at the expense of a skewed standard deviation. Such plots can therefore not be used to determine Km and Vmax by linear regression. Figures taken from [Buxbaum, 2007]. original data space

Lineweaver-Burk space 11

1

10 9 8

0.75

Vmax / v

v / Vmax

7

0.5

6 5 4 3

0.25 2 1 0

0 0

1

2

3

4

5 [S] / Km

6

7

8

9

10

-1

0

1

2

3

4

Km / [S]

The other two linearizations are those of Hanes: [S] [S] K = + m v Vmax Vmax

(7.5)

v [S]

(7.6)

and of Eadie-Hofstee: v = Vmax − Km ∗

They are rarely used because they lead to a mixing of dependent and independent variables, which causes problems when experimental results are evaluated by statistical methods. You may safely place them into passive memory. This is not to say that the Lineweaver-Burk-transformation were without such problems. On the contrary, taking of reciprocals transforms not only the data points, but also their standard deviation, which becomes not only unsymmetrical, but extremely large for low values of [S] (see fig. 7.3). The Lineweaver-Burk-transformation should therefore no longer be used to estimate Km and Vmax from experimental data. We have better procedures to do that by curve fitting directly to the original data on a computer (for experts: Nelson/Mead-simplex and Marquardt/Levenberg algorithms). However, as we will see later, the Lineweaver-Burk-transformation is still very useful for the presentation of the results of kinetic experiments.

7.3.2. Catalytic perfection k+1 , the rate of association between the enzyme and the substrate, is in aqueous solution limited to ≈ 1 × 109 M−1 s−1 due to the viscosity of water (note that this is a second order

144

Environmental influences on enzyme activity

7.3.3

Table 7.1.: Some enzymes approaching catalytic perfection. Note that some enzymes achieve a high efficiency by increasing kcat , others by decreasing Km . Data obtained from BRENDA. Enzyme Organism Substrate kcat Km kcat /Km s−1 M M−1 s−1 β-lactamase E. coli ampicillin 1090 8.0 × 10−5 8.7 × 108 Carbonic anhydrase Mus musculus CO2 940 000 1.6 × 10−3 5.9 × 108 Catalase N. crassa H2 O2 125 000 2.5 × 10−4 5.0 × 108 AcChE H. sapiens Ac-S-choline 6500 4.6 × 10−5 1.4 × 108 Peroxidase Strep. faecalis NADH 83.3 2.0 × 10−6 4.2 × 107 Fumarase S. scrofa fumarate 364 5.0 × 10−6 7.3 × 107 Triose-P isomerase S. cerevisiae d-GA3P 16 700 1.1 × 10−3 5.6 × 107 rate constant). Indeed, if we increase the viscosity of the solution by addition of agarose or other inert material, enzymatic reaction velocities are reduced. In the linear part we can rewrite the HMM-equation in the following way: v=

Vmax [S] k = cat [E][S] Km + [S] Km

(7.7)

where kcat /Km is called the efficiency constant of the enzyme. Note that it too, just like k+1 is a second order rate constant. k+1 is the rate at which substrate can bind to the enzyme, kcat /Km is the rate with which the enzyme can convert the substrate into released product. If the efficiency constant is of the same order of magnitude as k+1 , then the enzyme can handle the substrate as fast as it can be delivered into its binding site, and the enzyme is called catalytically perfect. Because the diffusion of the product of one enzyme to the substrate binding site of a second enzyme can be the rate limiting step of a biochemical pathway, enzymes forming a pathway may be arranged in complexes which directly pass intermediates between them. This eliminates not only the time required for an intermediate to diffuse, but reduces the risk of the intermediate undergoing other reactions, e.g. with water, before it can reach the next enzyme. Elucidating the interactome of cells has become an important research question.

7.3.3. Environmental influences on enzyme activity You have already seen that enzyme structure depends on environmental factors like temperature, pH, ionic strength or the presence of denaturing compounds. Of course the function

145

7.3.4

Biochemistry and Genetics

of enzyme depends on proper folding, so any of these factors will also affect the enzymatic activity. In addition, the following points are relevant: temperature higher temperatures mean more molecular motion and hence higher reaction rates. However, beyond a certain temperature the enzyme gets inactivated, hence enzymatic activity vs temperature is an optimum curve. Osmolarity Enzymes need water as a “grease” for conformational changes. Osmolytes reduce the available water concentration and hence the enzymatic activity. pH The protonation state of both the substrate and of amino acid R-groups in the catalytic center of the enzyme are influenced by pH.

7.3.4. Cooperativity As we have seen previously, interactions of substrates with many enzymes results in hyperbolic v vs. [S] curves. This is in particular the case when an enzyme has only one binding site for a substrate. If however there are several binding sites, binding of substrate to one site may change the conformation of the enzyme and hence its affinity for substrate at the other binding sites. This effect is called cooperativity, it results in S-shaped (sigmoidal) rather than hyperbolic binding curves. In addition to binding sites for additional substrate molecules (“homotropic effect”) there may also be binding sites for other molecules (“heterotropic effect”). Imagine a pathway leading from some substrate A to a product Z via intermediates B, C...Y. In such cases would it not be useful if the enzyme that catalyzes the conversion A * ) B (“Aase”) would be turned off by high concentrations of the end product Z, and turned on by low concentrations? This is called end product inhibition, and is achieved by heterotrophic allosteric regulation of enzyme activity. Phosphofructokinase, which catalyzes the first committed step in glycolysis, the breakdown of food glucose, is an example for this. Binding of a ligand to a receptor may also be regulated in such a fashion, e.g. oxygen binding to hemoglobin, which is regulated by [O2 ] (homotropically) and by pH (Bohr-effect) and 2,3-BPG (heterotropically). Cooperativity (using hemoglobin as a model) was first described by A. Hill in 1910. The equation looks very much like the HMM-equation, except that the substrate occurs as a power of h, the Hill-coefficient: v=

Vmax [S]h Km + [S]h

(7.8)

The Hill-coefficient has the following meaning: 0 ≤ h < 1 signifies negative cooperativity, where binding of a ligand to one site impedes the binding at other sites.

146

7.3.4

Cooperativity

Figure 7.4.: Top: Endproduct inhibition. In a biochemical pathway substrate A is turned into intermediate B by the enzyme Aase, B is turned into C by Base and so on. If the concentration of the final product Z is high, Z binds to Aase and inhibits it allosterically. At low concentrations of Z, Z is released from Aase and the activity of Aase increases. Bottom: This results in a allosteric effect on Aase, which has S-shaped (“sigmoidal”) v vs. [S] curves, the shape of the curve depends on [Z]. As a consequence the velocity of the reaction depends not only on [A], but also on [Z]. Figure taken from [Buxbaum, 2007].

Allosteric regulation of enzyme activity (Km and Vmax constant) 100

v (% of Vmax)

80

60 nH = 0.50 nH = 1.00 nH = 1.75 nH = 2.50 nH = 3.50

40

20

0 0

1

2 [S]/Km

3

4

147

7.3.4

Biochemistry and Genetics

Figure 7.5.: Top: Changes in the Hill-coefficient h affect the shape of the v/S-curve. h = 1 results in hyperbolic curves (no cooperativity, HMM-law), if h > 1 (positive cooperativity) then the curves are S-shaped (sigmoidal). If h < 1 (negative cooperativity) the curves are flatter than with enzymes following the HMMlaw. Bottom: Binding of regulators often does not change h but either K0.5 (K-type) or Vmax (V-type, much rarer). allosteric enzyme of K-type

1 h h h h h

v / Vmax

0.75

= = = = =

0.50 1.00 1.75 2.50 3.50

0.5

0.25

0

0

0.5

1

1.5 [S] / Km

2

2.5

Oxygen binding to HbA: Effect of 2,3-BPG

3

Allosteric enzyme of V-type

100 2 1.75 1.5

60 v / Vmax

Oxygen Saturation (%)

80

40

0.0 0.5 1.0 2.0 6.0

mM mM mM mM mM

allosteric inhibitor no effector allosteric activator

1.25 1 0.75 0.5

20

0.25

0

148

0

1

2

3 pO2 (kPa)

4

5

6

0

0

2

4

6 [S] / Km

8

10

7.3.4

Cooperativity

Figure 7.6.: Sequential model of cooperativity according to Koshland et al. The enzyme exists in a T-form (squares) with low and a R-form (circles) with high affinity for substrate. Empty molecules are white, molecules with bound substrate cyan. As more and more binding sites are filled, the probability of the protein to exist in the R-form increases. In the sequential model of Monod et al. the entire protein is either in the T- or in the R-form, i.e., only the outer two columns exist. all relaxed

all tense

L

L

L

L

L

L

L

L

L

L

L

L

L

L L

L L

L

L

L L

L

L L

L

L L

L L L

L

L L L L

L

L

L

L

L

L

L L

L

L

L

L

L L

L

L L

L

L

L

L L

L

L

L

L

L L L

L L

L

L L L

L L

149

7.4.1

Biochemistry and Genetics

h=1 means that there is no cooperation at all (binding follows HMM-kinetics) 1 < h < n positive cooperativity, binding of one ligand to one site facilitates binding to the other sites. h=n means complete cooperation (all sites are either filled or empty, no molecules with only some sites filled are allowed). h > n has never been observed, this situation is considered impossible on theoretical grounds.

7.4. Enzyme inhibition So far we have looked upon enzymes as totally specific, binding only their substrate or, at most, some allosteric regulator. If this were the case, you would not have to study pharmacology, for most pharmaceuticals work by binding to enzymes. Binding can be reversible, then we say the enzyme is inhibited. Removal of the inhibitor (e.g. by dialysis or gel filtration) restores enzyme activity. Alternatively, the interaction may be essentially irreversible, then we say the enzyme is inactivated (see next section). Depending on the mode of interaction between inhibitor, substrate and enzyme we distinguish several forms of inhibition. Note: The nomenclature was worked out by W. W. Cleland in 1963 (Biochim. Biophys. Acta 67 104-196), but is often misrepresented in textbooks. You may therefore have learned things differently in previous courses. The nomenclature represented here is however used by professional enzymologists and largely agrees with a proposed IUBMB standard.

7.4.1. Competitive inhibition In competitive inhibition substrate and inhibitor compete for the enzyme, i.e., they can not bind at the same time: S

E

kcat Ks

I

ES

E

+

P

competitive inhibition

Ki EI

From a competitive inhibition mechanism it has often been concluded that substrate and S inhibitor share the same binding kcat site on the enzyme. This however is incorrect, if substrate binding ofE the in such a way that the inhibitor can no ES E changes the conformation P + enzyme inhibition Ks vice versa then the inhibitionuncompetitive longer bind and mechanism will also be competitive: I Kii

EIS

150 S E

Ks

I Ki EI

Kss

kcat

ES Kii EIS

E I

+

P

non-competitive inhibition

7.4.2

Uncompetitive inhibition

S

00 11 00 11 00 11 I 00 11

S

P

00 11 00 11 00 11 I 00 11 Enzyme

S

00 11 00 11 00 11 I 00 11

00 11 00 11 00 11 I 00 11 S

Enzyme

Enzyme

Enzyme

S P S

Enzyme

Enzyme

Enzyme

I

I

I

I

Of course, if substrate and inhibitor share the same binding site, then the inhibition mechanism must be competitive, as no 2 objects can occupy the same space at the same time. Because high [S] can exclude the inhibitor from binding to the enzyme the effect of any given [I] can be counteracted by increasing [S]. However, as the [I] increases, higher and higher [S] are required to achieve a given v. Thus Vmax is not changed in competitive inhibition (see fig. 7.7). In the Lineweaver-Burk-plot all lines intersect at a common point on the y-axis, that is, at a common 1/Vmax . If one plots the slopes of the lines in the Lineweaver-Burk-plot as a function of [I] (not its reciprocal!) the data points are on a straight line, Ki can be determined from its intersection with the x-axis (secondary plot).

Example for the medical use of competitive inhibition

Ingested methanol is oxidized by alcohol dehydrogenase to methanal (formaldehyde), which is highly toxic to the optic nerve, leading to blindness. Ethanol inhibits methanol oxidation competitively and hence protects the optic nerve from damage. Ethanol concentration has to be kept very high (close to lethal) for several days, until the methanol has been excreted by the kidneys. Important pharmaceuticals like methotrexate or sulphonamides also act as competitive inhibitors, these will be discussed with the enzymes on which they act.

151

7.4.2

Biochemistry and Genetics

Figure 7.7.: Competitive inhibition. Top: plot of v vs [S] at several [I]. Bottom: Lineweaver-Burk-transformation and secondary plot. Figures taken from [Buxbaum, 2007]. competitive inhibition no inhibition [I] = 0.5 * Ki [I] = 1 * Ki [I] = 2 * Ki [I] = 4 * Ki [I] = 6 * Ki [I] = 10 * Ki

1

v / Vmax

0.75

0.5

0.25

0

0

1

2

3

4

5

6 [S] / Km

7

8

Lineweaver-Burk-plot, competitive inhibition

9

10

11

12

secondary plot, competitive inhibition

10

12

9 10 slope in Lineweaver-Burk-plot

8 7

1 / v

6 5 4

no inhibition [I] = 0.5 * Ki [I] = 1 * Ki [I] = 2 * Ki [I] = 4 * Ki [I] = 6 * Ki [I] = 10 * Ki

3 2

8

6

4

2

1 0

152

-1

0

1

2

3

4 1 / [S]

5

6

7

8

9

0

-Ki

0

2

4 [I]/Ki

6

8

10

7.4.2

Uncompetitive inhibition

Figure 7.8.: Uncompetitive inhibition. Top: plot of v vs [S] at several [I]. Bottom: Lineweaver-Burk-transformation and secondary plot. Figures taken from [Buxbaum, 2007]. uncompetitive inhibition no inhibition 1 [I] = 0.5 * Kii [I] = 1 * Kii [I] = 2 * Kii [I] = 4 * Kii [I] = 6 * Kii [I] = 10 * Kii

v / Vmax

0.75

0.5

0.25

0

0

1

2

3

4

5 [S] / Km

6

7

Lineweaver-Burk-plot, uncompetitive inhibition

8

9

10

secondary plot, uncompetitive inhibition 12

y-intercept in Lineweaver-Burk-plot

18

15

1 / v

12

9 no inhibition [I] = 0.5 * Ki [I] = 1 * Ki [I] = 2 * Ki [I] = 4 * Ki [I] = 6 * Ki [I] = 10 * Ki

6

3

0

-1

0

1

2

3

4 1 / [S]

5

6

7

8

9

10

8

6

4

2

0

-Kii

0

2

4 [I]/Kii

6

8

10

153

S

7.4.3 E I

kcat

Biochemistry and Genetics

ES

Ks

E

+

P

7.4.2. Uncompetitive inhibition

competitive inhibition

Ki

In uncompetitive inhibition the inhibitor reacts exclusively with the enzyme-substrate comEI plex, not with the free enzyme: S kcat ES E E + P uncompetitive inhibition Ks I Kii EIS

If we increase [S], we increase [ES] and this will lead to higher [ESI]. Increasing [I] will S pull ES into ESI, following Le kcatChatelier’s principle more E must be converted to ES to maintain the equilibrium constant K . As a result, the inhibitor increases the apparent E ES E s+ P affinity of the versa. non-competitive inhibition Ks enzyme forI the substrate and vice I Kii Ki Since we can not combat the effect of inhibitor by increasing [S], Vmax must decrease K S ss with increasing [I] (see fig. 7.8). Because the intersections on the y- and x- axis in the kcat EIS EI Lineweaver-Burk-plot are the reciprocals of K and V , respectively, both increase (go

P further away from 0) with increasing E[I].+Characteristic uncompetitive inhibition are the competitivefor inhibition S K I s parallel lines in the Lineweaver-Burk-plot. Plotting the y-intercepts of the LineweaverKi against [I] in a secondary plot gives a straight line, intersecting the x-axis at Burk-plot −Kii . S EI kcat E ES E + P that the enzyme acts as an oligomer. Uncompetitive inhibition SK inhibition is rare and is a sign mixed I s I k cat Kii K ES Ei E + P k* uncompetitive inhibition Kss Ks I inhibition 7.4.3. Noncompetitive EIS EI P EI + K E

m

ES

max

ii

S In noncompetitive inhibition both the free enzyme and the enzyme-substrate complex can EIS bind the inhibitor: S E

Ks

I Ki

Kss

EI

kcat

ES

E

+

P

I

Kii

non-competitive inhibition

EIS

S

The four dissociation constants are related by the “law of micro-reversibility”, which S

154 I

E

Ks Ki Kss

EI S

kcat

ES Kii EIS

E

+

P

EI

+

P

I k*

mixed inhibition

155 0

2

4

6

8

10

12

-2

0

2

4

6

8

10

12

14

16

18

20

-3

-2

Ki

-2

0

0.25

0.5

0.75

0

0

0

y-intercept slope

Kii

1

2

3 4 1 / [S]

5

6

7

2

4 [I]

6

8

secondary plot, non-competitive inhibition (Ki > Kii)

-1

no inhibition [I] = 0.5 * Ki [I] = 1 * Ki [I] = 2 * Ki [I] = 4 * Ki [I] = 6 * Ki [I] = 10 * Ki

8

Lineweaver-Burk-plot, non-competitive inhibition (Ki > Kii)

v / Vmax

1

10

9

1

0

2

4

6

8

10

12

-2

0

2

4

6

8

10

12

14

16

18

20

-2

Ki, Kii

-3

2

no inhibition [I] = 0.5 * Ki [I] = 1 * Ki [I] = 2 * Ki [I] = 4 * Ki [I] = 6 * Ki [I] = 10 * Ki

1 / v slope and y-intercept in Lineweaver-Burk-plot

4

5 [S] / Km

6

7

0

0

1

2

3 4 1 / [S]

5

6

7

2

4 [I]/Ki

6

8

secondary plot, non-competitive inhibition (Ki = Kii)

-1

no inhibition [I] = 0.5 * Ki [I] = 1 * Ki [I] = 2 * Ki [I] = 4 * Ki [I] = 6 * Ki [I] = 10 * Ki

8

Lineweaver-Burk-plot, non-competitive inhibition (Ki = Kii)

3

non-competitive inhibition Ki = Kii

10

9

8

slope and y-intercept in Lineweaver-Burk-plot

1 / v

slope and y-intercept in Lineweaver-Burk-plot

1 / v

0

2

4

6

8

10

12

-2

0

2

4

6

8

10

12

14

16

18

20

-3

-2

-2

Kii

9

0

0

slope y-intercept

Ki

1

2

3 4 1 / [S]

5

6

7

2

4 [I]

6

8

secondary plot, non-competitive inhibition (Ki < Kii)

-1

no inhibition [I] = 0.5 * Ki [I] = 1 * Ki [I] = 2 * Ki [I] = 4 * Ki [I] = 6 * Ki [I] = 10 * Ki

8

Lineweaver-Burk-plot, non-competitive inhibition (Ki < Kii)

10

10

9

Figure 7.9.: Noncompetitive inhibition. Top: plot of v vs [S] at several [I]. Bottom: Lineweaver-Burk-transformation and secondary plot for the three possible cases, Ki > Kii , Ki = Kii and Ki < Kii . Figures taken from [Buxbaum, 2007].

Noncompetitive inhibition

7.4.3

7.4.3

Biochemistry and Genetics

can be derived from the definition of the constants: K Ki = s Kii Kss

(7.9)

What does this mean? Lets look at the three possible cases in turn: Ki = Kii This means that the affinity of the enzyme for the inhibitor is independent of whether or not it has substrate bound. In other words, substrate binding does not change the conformation of the enzyme in such a way that the affinity for the inhibitor would be changed. If this is the case however, then the opposite must also be true: Ks = Kss . In the Lineweaver-Burk-plot all lines intersect in a common point on the x-axis, because Ks and Km are related and if Ks is not changed by inhibitor binding, Km can’t be either. Ki > Kii In this case the affinity of E for the inhibitor is smaller than the affinity of ES (remember that dissociation constants are reciprocals of the affinity!). In other words, binding of substrate changes the conformation of the enzyme so that binding of the inhibitor is facilitated. This however must also mean that the affinity of EI for the substrate is higher than that of E, binding of I increases the affinity and lowers the dissociation constant (and hence the apparent Km ). Ki < Kii This is the opposite case from above, binding of substrate changes the conformation of the enzyme in such a way that binding of the inhibitor is made more difficult. Then in turn binding of the inhibitor must change the enzyme conformation so that binding of the substrate becomes more difficult, the dissociation constant (and hence the apparent Km ) is increased. Ki and Kii are determined from secondary plots, Kss is then calculated from the law of micro-reversibility. H3C

C O

H N

O S N N

S O

NH2

A pharmaceutical working by non-competitive inhibition is acetazolamide (Dianox), which binds to an essential Zn2+ ion in carbonic anhydrase. The + enzyme catalyzes the reaction CO2 + H2 O * ) HCO− 3 + H , for example on the luminal membranes of kidney tubule cells. The bicarbonate in the primary urine is broken down to carbon dioxide, which diffuses through the membrane into the cell and then into the blood. If carbonic anhydrase is inhibited, more bicarbonate ions stay in the urine, Na+ and K+ follow for electro-neutrality and water osmotically. As a result urine volume is increased (diuresis). By a similar mechanism the drug is effective in treating increased intraoccular pressure (glaucoma) and increased intra-cranial pressure (resulting in absence seizures). Currently it is also on clinical trials for acute mountain sickness which may befall people at great heights where the air pressure is reduced and hence the amount of oxygen in a

156

S E

Ks

kcat

ES I

Kii

E

+

P

uncompetitive inhibition

Enzyme inactivation

7.5

EIS

given volume of air. The hypoxemia results in faster breathing (hyperventilation), this in turn to aS loss of carbon dioxide resulting in alkalosis. Inhibition of carbonic anhydrase slows the decomposition of bicarbonate into carbon dioxide and hence helps to stabilize kcat blood EpH. ES E + P Ks

I

Ki

non-competitive inhibition

I

Kii

Kss inhibition 7.4.4. Mixed EIS

EI

is similar toS the noncompetitive, except that the EIS-complex has some remaining enzymatic activity: S E

Ks

I Ki

Kss

EI

kcat

ES Kii

E

+

P

EI

+

P

I k*

EIS

mixed inhibition

S

The Lineweaver-Burk-plot also looks similar to the noncompetitive case (all three possibilities exist too), but the secondary diagram is curved. This allows mixed and noncompetitive inhibition to be easily distinguished. Mixed inhibition is rare in practice, pharmacologically speaking it would be useless: If we need to inhibit an enzyme to help a patient, we don’t want it to retain activity after binding the inhibitor! Note however that mixed inhibition is the most universal mechanism of inhibition, by simply setting some rate constants to 0 we can get any of the other mechanisms from it. In physiology however you will learn about partial (ant)agonists for receptors.

7.5. Enzyme inactivation From the medical point of view, inhibitors have a big disadvantage: Because their interaction with enzymes is reversible their effect diminishes as the inhibitor is excreted or metabolized. Inactivators on the other hand completely blow the enzyme to kingdom come, making them much more attractive pharmaceuticals. Inactivation often, but not necessarily, involves covalent modification of the enzyme. There are two classes of inactivators: unspecific destroy protein structure. Acids or heavy metal salts belong into this group. Obviously one would not normally use them pharmaceutically.

157

7.5

Biochemistry and Genetics

Figure 7.10.: Mixed inhibition. Top: plot of v vs [S] at several [I]. Bottom: LineweaverBurk-transformation and secondary plot. Figures taken from [Buxbaum, 2007]. mixed non-competitive inhibition V2 = 1/2 V, Ki = Kii

1

v / Vmax

0.75

f(x)

0.5 no inhibition [I] = 0.5 * Ki [I] = 1 * Ki [I] = 2 * Ki [I] = 4 * Ki [I] = 6 * Ki [I] = 10 * Ki

0.25

0 0

1

2

3

4

5

6 [S] / Km

7

8

Lineweaver-Burk-plot, mixed non-competitive inhibition V2 = 1/2 V, Ki = Kii

9

10

11

12

secondary plot, mixed non-competitive inhibition V2 = 1/2 V, Ki = Kii

10

2

9 8 1.5

7

slope

1/v

6 5 4

no inhibition I = 0.5 Ki I = 1.0 Ki I = 2.0 Ki I = 4.0 Ki I = 6.0 Ki I = 10.0 Ki

3 2

1

0.5

1 0

0 -1

158

0

1

2

3

4 1 / [S]

5

6

7

8

9

-1

0

1

2

3

4

5 [I]/Ki

6

7

8

9

10

7.6

Enzyme inactivation

Figure 7.11.: Lactam-antibiotics work by inactivation of transpeptidase, an enzyme required for bacterial cell wall synthesis. O

H N O

H S

CH3

Penicillin

+

CH3

N

HO

COO-

Ser-Enzyme

Transpeptidase

-

COO CH3 CH3 O C H2

C

S

C

N

H

O C N H C H O

Ser-Enzyme

Inactivated enzyme

specific these compounds interact with one protein only, the protein can be protected by the presence of its substrate. A special case in the latter group are the suicide substrates, which bind to the substrate site of an enzyme and are there converted into the active species by the enzymes own k+2 k+1 GGGGGGGB catalytic activity: E + I F E · I G G G GGGGAE−I. Since both the binding site and the enzyGG k−1 matic activity are required, suicide substrates are very specific for their target and often have little side effects. The reaction velocity of suicide inactivation is determined by slow conversion of first complex, this is a first order reaction: v = k+2 [E · I] = −d[E]/dt

(7.10)

Integration yields [E]t = [E]0 ∗ e−k+2 ∗t

→

ln([E]t ) = ln([E]0 ) − k+2 ∗ t

(7.11)

Thus if one plots the logarithm of remaining activity as a function of time, one gets a straight line. If the experiment is repeated with different inactivator concentrations, one can plot the slopes of the lines as a function of [I], this results in a hyperbola, simply because the conversion of E · I into E−I requires time. The rational is the same as with the HMM-equation (see fig. 7.13).

159

7.6

Biochemistry and Genetics

Figure 7.12.: Aspirin inactivates Cox-1 by Ser-acetylation, it also competitively inhibits Cox-2. In platelets no re-synthesis of Cox-1, thus coagulation is prevented for the entire life time of the platelet! O C

O

O C

OH

OH

C

OH

OH

O C CH3 O Acetyl salicylate (aspirin®)

salicylic acid

O CH3

Methyl salicylate (oil of wintergreen)

Figure 7.13.: Inactivation of an enzyme by a suicide substrate. For details see text. Figures taken from [Buxbaum, 2007]. time course concentration dependence

100 [I]/Ki = 0.1 = 0.2 = 0.5 = 1.0 = 2.0 = 5.0 = 10 normalised rate constant k / kmax

1

[E] (% of [E]0)

10

1

0.75

0.5

0.25

0.1

0

0

160

5

10

15

20

25 30 time (rel. units)

35

40

45

50

0

2

4 6 normalised inactivator conc. [I] / Ki

8

10

7.6

Enzymes with multiple substrates or products

7.6. Enzymes with multiple substrates or products So far we have looked at enzymes with one substrate and one product. If this were all, biochemistry would be kind of boring. To write down the mechanism of reactions with multiple substrates or products Cleland has introduced a simple notation. Lets look at the single substrate reaction (E + S * ) ES * ) EP * ) E + P) first:

S

P

( ES EP ) E

E

The reaction is drawn at a long horizontal arrow, with vertical arrows denoting the binding of substrates or dissociation of products. Reactions inside the enzyme — without entering or leaving compounds — are written in brackets, these are called inner complex. If an enzyme uses two substrates it may be irrelevant which of them binds first, this is called an random bi mechanism: S1

P2

S2

ES1

(

S2ES1 P2EP1

P1

EP1

)

E

E

S2E

S2

P2E

S1

P1

P2

The same of course may also be true for two products. On the other hand, the enzyme may also require that one particular substrate binds first, only the conformational changes associated with that binding open the second substrate binding site:

S1

E

S2 ES1

P

(

S2ES1 EP

) E

This is called an ordered bi mechanism, here drawn with a single product.

161

7.7

Biochemistry and Genetics

The third possibility is the so called ping pong mechanism, where the first substrate binds, transfers a functional group to the enzyme (or to a prosthetic group) and then leaves as the first product. Only then is the second substrate bound, accepts the functional group and leaves as the second product: S2 P2-X S1-X P1

X-ES2

ES1-X

( EP -X )

( X-EP )

X-E 1 2 E E Many transferases work according to this mechanism, catalase (2H2 O2 → 2H2 O + O2 ) is another example: H2O

H2O2

(

3+

H2O2

5+

O H2O

E-Fe

E-Fe

)

5+

E-Fe

.

4+

E-Fe

O

H2O2

O

3+

E-Fe

(

. E-Fe

H2O

4+

O

3+

O2 H2O

E-Fe

H2O2

)

O2 3+

E-Fe

O2 3+

E-Fe

Note the ordered release of the last products! Enzymes with more than two substrates work in the same way, instead of bi- one would use ter-, quad- and so on.

7.7. How do enzymes do it? There are a only a few fundamental mechanisms by which enzymes achieve acceleration of reactions: Orientation Reactants are bound by the enzyme in a spatial orientation that facilitates reaction. Reduction of reaction order Reactants are held together by the enzyme much longer than in random collisions. Reactions of n-th order between molecules become 1st order reaction inside a molecule. Acid/base catalysis Unfavorable charge development on intermediate is prevented by donation of a proton from H3 O+ (specific acid catalysis), HA (general acid catalysis) or its abstraction by OH− (specific base catalysis) or B : (general base catalysis). pKa values of amino acid side-chains are adjusted by the environment inside the protein to facilitate the reaction. Example: Glu pKa in xylanase is between 4.6 and 6.7. Redox-reactions on cofactors Redox-potential is again adjusted by protein environment, e.g. Fe3+ + e− → Fe2+ has E 00 of −432 to 771 mV.

162

7.7.1

Protease reaction mechanism

Electrostatic catalysis an electrical charge on the intermediate can be stabilized by a nearby opposite charge. Example: Zn-containing dehydrogenases. Electrical field strength inside proteins can be in the order of 10 MV/cm, separation of 1 unit charge over 1 Å lowers ∆E a by 9.6 kJ/mol. Rack-mechanism Bonds of the substrate are strained during formation of the ES‡ intermediate. Quantum theory Electrons or protons tunnel through the energy barrier, rather than crossing it. The classical model of substrate binding, the lock-and-key model, was introduced by E. Fischer in 1894. In this model the substrate fits into a preformed binding site within the enzyme. However, this would actually increase the activation energy rather than reducing it:

ΔEa

ES |

free energy G

free energy G

S

lock-and-key mechanism

|

EP

E+S

induced fit mechanism

free energy G

without catalyst

ΔEa

ΔGB ES ES

|

EP

ΔEa

E+S

S ΔG

ΔG E+P

P

ΔGB

ΔG E+P

ES

Reaction progress

Reaction progress

Reaction progress

In 1959 D.E. Koshland proposes the induced fit hypothesis to solve this problem. In this model the enzyme binds not the substrate, but the transition state. Thus part of the energy required to convert the substrate into the transition state is compensated by the binding energy to the enzyme, the activation energy is the difference between activation and binding energy, rather than its sum as in the lock and key model. According to the induced fit hypothesis the binding site in the enzyme is not preformed, but rather the enzyme adapts itself to the transition state as this is formed. Application: Antibodies against a transition state analogue may have catalytic properties (catalytic antibodies).

7.7.1. Protease reaction mechanism Peptides and proteins are stable, because during their hydrolysis a high-energy intermediate is formed, which converts back to the original compound much easier than to the next intermediate:

163

R´

H N H R

X O C O

+

R´

+

H N H

R

X O C O

R´

H N H

R

+

H

X O

X O C O

R´

C O

N H

Biochemistry and Genetics

+

R

7.7.1

Acids and bases can accelerate the conversion of the first into the second, more stable intermediate and hence peptide bond splitting, that is why they are caustic to our skin! Rather than working with extreme pH, proteases in our body stabilize the transition state by distributing the charges onto nearby amino acids, as shown here for Ser-proteases: 57

His H N 102

Asp

C

195

Ser

O:

+

C O

Enzyme-substate complex

O

H

H N Ser

N

Ser

N

+

O C O H N H R

O

H N Gly

H

H N Gly

H

Transition state 1

Asp

C

N

Free enzyme

N

O O

R

O

C O

O

OH

His Ser H C

His Ser

H N Ser

R

Asp

193

H N Gly

R

N

O

His

C

H N Ser

N H

H

R

Asp

195

R

+

N

O C O H

N

R

H N Ser

N H

His Ser

H N Gly

OH H

R

O

Asp

O

H N Ser

O C O

C

H

+

N

N

O

H N Gly

H

Transition state 2

O

N H R R His Ser H Asp

C

N

N

H N Ser

O C O

R

H N Gly

His Ser

Acyl-enzyme intermediate

O

H

O

Asp

C

N

N

H N Ser

O C O H

O

H N Gly

H

O O

H2O

Ser-195 (rather than water) acts as a nucleophile and forms the intermediate. The positive

164

Adenosine Triphosphate (ATP)

7.8.1

charge is immediately transferred to His-57, there it is stabilized by salt bond formation with Asp-102 (catalytic triad). The negative charge of the intermediate is stabilized by hydrogen bonds to the amide nitrogens of Ser-195 and Gly-193 (oxanion hole). In a second step the His-57 transfers a hydrogen onto the nitrogen of the peptide bond, allowing the first product to dissociate. The resulting Acyl-enzyme intermediate is then hydrolyzed by a similar mechanism.

7.8. Coenzymes Some (not all) enzymes require a coenzyme for their reaction. There are 2 types of coenzymes: 1. Some coenzymes are bound permanently to the active site of the enzyme, either covalently or noncovalently. These coenzymes are called prosthetic groups. 2. Some coenzymes are soluble molecules which associate with the enzyme active site only during the reaction. They function as co-substrates. Like other substrates, they become changed chemically during the reaction and have to be regenerated in a different reaction.

7.8.1. Adenosine Triphosphate (ATP) ATP is a co-substrate in reactions of energy metabolism. Chemically, it is a nucleotide: NH2 N O O O H2 O P O P O P O C

-

O

O

N N

Adenine

N

O

O Ribose

Polyphosphate

OH

OH

ATP can serve as the energetic currency of the cell because it contains 2 energy-rich phosphoanhydride bonds, each with a free energy content of 30.5 kJ/mol. ATP is synthesized from ADP + phosphate during catabolic reactions, most of this by oxidative phosphorylation in the mitochondria. ATP hydrolysis drives: • Biosynthetic (anabolic) reactions

165

7.8.1

Biochemistry and Genetics

• Active transport across membranes • Cell motility and muscle contraction There are 2 modes of ATP hydrolysis: 1. ATP + H2 O → ADP + Phosphate 2. ATP + H2 O → AMP + Pyrophosphate In the second mode the pyrophosphate (PPi ) is quickly hydrolyzed by soluble pyrophosphatases, making the reaction irreversible at the expense of an energy rich bond. ATP is used for: • Nucleic acid synthesis. ATP is an immediate precursor for RNA synthesis and an indirect precursor for DNA synthesis. • Phosphorylation reactions. ATP can transfer its last phosphate to an acceptor molecule. These reactions are catalyzed by kinases, a type of transferase. Example: H2C OH

H2C OH HO CH

+

ATP

C OH H2 Glycerol

HO CH

O

+

ADP

C O P O H2 O Glycerol-3-phosphate

• Coupling to endergonic reactions. Endergonic reactions cannot proceed in the cell unless the enzyme couples the endergonic reaction to ATP hydrolysis. Example: Palmitic acid + CoA−SH → Palmitoyl-CoA + H2 O, ∆G00 = +32.8 kJ/mol. This reaction cannot proceed in the indicated direction. The reaction that actually takes place in the body is: Palmitic acid + CoA−SH + ATP → Palmitoyl-CoA + AMP + Pyrophosphate ∆G00 = +2.8 kJ/mol. This reaction proceeds in the indicated direction because its ∆G00 is close to zero and one of the products (pyrophosphate) is rapidly removed from the equilibrium. Important for the reaction equilibrium: • The reaction proceeds whenever the actual ∆G (not the ∆G00 !) is negative. Therefore the reaction can be driven in the right direction when the concentration of a substrate is very high and/or that of a product is very low. In the above example, pyrophosphate is very low. Also, the [ATP] / [AMP] ratio in the cell is about 100, and this lowers the real ∆G by about 11.3 kJ/mol. • When ATP is hydrolyzed in the reaction, the ∆G00 is reduced by 30.5 kJ/mol. This is usually sufficient to drive the reaction.

166

7.8.2

Redox Coenzymes

In the cell, the adenine nucleotides are in equilibrium through the adenylate kinase (myokinase) reaction: ATP + AMP * ) 2 ADP.

The energy status of the cell can be described by the cellular [ATP] /[ADP] ratio, or by the energy charge: ν=

[AT P ] + 1/2[ADP ] [AT P ] + [ADP ] + [AM P ]

(7.12)

The nucleotides guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP) are also used as coenzymes in some energy-dependent reactions.

7.8.2. Redox Coenzymes

Some coenzymes transfer either naked electrons or electrons + protons (hydrogen atoms) during redox reactions. Nicotinamide-adenine dinucleotide (NAD+ ) and its phosphorylated derivative NADP+ are cosubstrates in dehydrogenase reactions. Structure: NH2 N

N N

N

H

O NH2

O

H2 O H2C O P O P O C O

+

O

H O

H

Nicotinamide

Adenine

N

.. +

-

H ,2 e

O

NH2

N R

O Ribose

Ribose

OH

OH

OH

OH

The flavin nucleotides flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD) are also hydrogen carriers, but they function as tightly bound prosthetic groups of the flavoproteins. Structure of FAD:

167

7.8.3

Biochemistry and Genetics O

Adenine

H3C

N

H3C

N

HC OH

N N

N

CH2

NH2

N

NH O Flavin Ribitol

} Riboflavin

HC OH O

O

HC OH

N O H2C O P O P O CH2 O O

O

H +

-

H+e

H3C

N

H3C

N

C

NH N

O

R Ribose

OH

OH

FADH, semiquinone radical

Flavin adenine dinucleotide (FAD), oxidised (FMN = Riboflavin + 1 Phosphate) +

-

H+e

H

O

H3C

N

H3C

N

N

R

H

NH O

FADH2, fully reduced

Straight electrons (without protons) can be transferred by many iron-containing proteins. In the iron-sulfur proteins the iron is bound to cysteine side chains, and in the heme proteins it is part of the heme group. The iron switches between the ferrous (Fe2+ ) and the ferric (Fe3+ ) state during the electron transfer. Other metal ions are also found in some enzymes, including zinc, copper and manganese. In some enzymes the metal ion participates in electron transfers, but in others it acts as a Lewis acid (electron pair acceptor).

7.8.3. Other Coenzymes There are many other coenzymes, each with its own special function. Examples:

Coenzyme A (CoA) is a cosubstrate. It contains a sulfhydryl group which forms thioester bonds with organic acids. Example: GTP CoA SH

168

+

H3C COO

-

GMP PPi

O CoA S C CH3

7.9

Enzymes in clinical diagnostics

Spectrum of NAD and NADH (0.5 mM in water) 2.5

NAD NADH

Absorbance

2

1.5

height: proportional to substrate concentration

absorbance 345 nm

Figure 7.14.: The optical test of Warburg is based on the different absorbance of NAD(P) and NAD(P)H at 350 nm. This allows both substrate concentrations and enzyme activities to be determined. Figures taken from [Buxbaum, 2007].

slope: proportional to enyzme activity

1

0.5

time 0 250

300

350 wavelength (nm)

400

sample was added here

S-Adenosylmethionine (SAM) is a cosubstrate that contains a methyl group. This methyl group is transferred to an acceptor during enzymatic reactions. Many, but not all, coenzymes contain a vitamin as part of their structure: Nicotinamide (niacin) in NAD+ and NADP+ , ribloflavin in FMN and FAD, pantothenic acid in coenzyme A.

7.9. Enzymes in clinical diagnostics If cells are damaged, the cell content, including the enzymes present inside the cell, are released into the blood stream. Because each enzyme molecule can turn over many, many substrate molecules, enzyme activities present in serum (the liquid obtained by letting blood coagulate) can be detected with high sensitivity and specificity. Since different organs have different functions, they also have different enzymes, and even if two organs have the same enzymatic activity, it may be carried out by different isoenzymes. Therefore, it is possible to test for organ damage by testing for appropriate enzymatic activities in a blood sample, a cheap, simple and relatively non-invasive technique. This is important for example for the diagnosis of myocardial infarct, liver and pancreas damage, muscular dystrophy and some cancers (see chapter 10 on page 203 for details).

169

7.10.1

Biochemistry and Genetics

Very commonly used is the optical test of Warburg, which is based on the fact that the nicotinamide group of NAD+ or NADP+ does not absorb at 350 nm in the oxidized state, upon reduction however it does (see fig. 7.14). The difference between the absorbance at the beginning and the end of the reaction is proportional to the substrate concentration, the rate of absorbance change is proportional to the enzyme concentration. Many enzymes however do not use NAD(P) in their reaction. For example creatine kinase performs the following reaction in muscle cells: Creatine kinase GGGGGGGGGGGGGGGGGGGGGB Creatine + ATP F GG Creatinephosphate + ADP. The enzyme is released into the blood stream after muscle damage, especially after an acute myocardial infarct. Thus if a patient reports to you with chest pain (angina pectoris) you can measure the activity of creatine kinase to determine whether or not the patient has an infarct. The reaction itself however does not lead to a change in absorbance. Therefore it is coupled with two other reactions: Pyruvate kinase GGGGGGGGGGGGGGGGGGGGGB ADP + Phosphoenolpyruvate F GG Pyruvate + ATP and

Lactate dehydrogenase GGGGGGGGGGGGGGGGGGGGGGGGGGGGGB Pyruvate + NADH + H+ F GG Lactate + NAD+ . Thus each molecule of ADP produced in the first reaction leads to the production of exactly one molecule of NADH + H+ in the third. The reagents are sold premixed by supply companies, only a serum or plasma sample needs to be added.

7.10. Membrane Transport Regulated transport across membranes is required for the exchange of nutrients, metabolic intermediates, and waste products; to maintain ion gradients; and to transfer nutrients, electrolytes, and water across absorptive epithelia.

7.10.1. Passive transport Passive transport does not require an energy source and is always down the electrochemical gradient. It includes passive diffusion (no carrier involved) and facilitated diffusion (through a carrier). Passive diffusion across the lipid bilayer depends on the physical properties (size, lipid solubility) of the diffusing molecule. No energy required. Transport rate depends linearly on substrate concentration. Examples for passive diffusion:

170

7.10.1

Passive transport

Figure 7.15.: Modes of membrane transport. For details see text. Figure taken from [Buxbaum, 2007].

H

O2

H

ADP + Pi 2K

• Most membranes are relatively permeable to water, although there are also water channels in some cells. • Osmosis is the passive diffusion of water from a compartment with lower solute concentration to one with higher solute concentration. • Biological membranes are permeable to various extents to some very small hydrophilic molecules: urea, methanol, ethanol. • Blood gases (O2 , N2 , CO2 ) are permeable, but the bicarbonate ion is not. • Membranes are permeable for small lipid-soluble substances (fatty acids, steroid hormones, some drugs). If the molecule is ionizable, only the uncharged form diffuses.

Carrier-mediated transport (facilitated diffusion, uniport) requires an integral membrane protein (“carrier” that interacts with the solute. Carrier-mediated transport shows: • Substrate specificity • Saturability (hyperbolic relationship between rate and concentration) • Specific inhibition and regulation

171

7.10.2

Biochemistry and Genetics

The carrier forms a gated channel that undergoes a conformational change during the transport cycle. Examples of Facilitated Diffusion • Glucose uptake by liver, erythrocytes, etc. is effected by an electroneutral uniport carrier. • The blood-brain barrier transport of glucose and amino acids.

7.10.2. Active transport Active transport is always carrier-mediated. It can accumulate a solute against its electrochemical gradient. Primary active transport hydrolyzes ATP while secondary active transport transports a second solute down its electrochemical gradient (Co-transport): The carrier transports at least two different solutes, either as Symport Two solutes transported in the same direction. Antiport Two solutes transported in opposite directions. either as Electroneutral transport:]No net transport of electrical charges. Electrogenic transport: Net transport of electrical charges. Examples of co-transport Symport Sodium cotransport of amino acids and glucose in the intestinal mucosa and the renal tubules, and of amino acid neurotransmitters into the nerve terminal. Antiport (=“exchange diffusion”) Sodium-calcium exchange across plasma membrane of many cells. Shuttles in the inner mitochondrial membrane. primary active transport in mammals involves ATP-hydrolysis. In plants and bacteria other energy rich compounds may be used to fuel the reaction (e.g. phosphoenol pyruvate, pyrophosphate). Na+ /K+ -ATPase (sodium-potassium ATPase) is in the plasma membrane of all cells with highest activity in brain and muscle. The sodium-potassium ATPase is an electrogenic antiporter, with 3 sodium pumped out of the cell and 2 potassium pumped into the cell. The transport cycle requires the phosphorylation of an aspartate side chain in the carrier by ATP. It transports Na+ out of and K+ into the cell. Membrane ATPases consume 10–30 % of the basal metabolic rate. Cardiotonic steroids inhibit the sodium-potassium ATPase, thereby reducing

172

Ion concentrations

7.11.1

the sodium gradient across the plasma membrane. This impairs the sodiumdependent calcium-extrusion mechanism. Increased intracellular calcium leads to an increased force of contraction of the heart (positive inotropic effect). Ca2+ -ATPase is present in the sarcoplasmic reticulum of muscle fibers and in the plasma membrane of many cells. It pumps calcium out of the cytoplasm and works by a mechanism similar to that of the Na+ /K+ - ATPase.

7.11. Homeostasis of the Intracellular Environment Cellular processes depend on temperature, pH and in many cases, the correct concentrations of inorganic ions. Differences between intra-and extracellular environment:

7.11.1. Ion concentrations Ion Na+ K+ Mg2+ Ca2+ Cl− HPO2− 4 HCO− 3

Extracellular (mM) 140.0 4.7 1.4 2.5 113.0 2.0 2.8

Cytoplasmic (mM) 10.0 140.0 30.0 0.001 4.0 11.0 10.0

The calcium concentration is very low in the cytoplasm but much higher in mitochondria and ER. Phosphate is high in the cell because of its role in energy metabolism (ATP synthesis!). The transmembrane gradients of sodium and potassium are required for cell excitability. The pH is about 7.4 in blood plasma and extracellular fluid, but near-neutral in the cytoplasm: pH = 6.8 at 37 °C, pH = 7.0 at 25 °C. The pH difference across the plasma membrane favors the transfer of carbon dioxide out of the cell, and it facilitates the removal of acidic products such as lactic acid. As far as possible, reducing conditions are maintained inside the cell. This is needed to protect proteins, lipids, and nucleic acids from oxidative damage. Most of the cellular metabolites are charged at pH 7. This is because the important acidic groups (carboxy and phosphate) have pKa s below 7, and the important basic groups (amino groups) have pKa s above 7. It means that most metabolites require carriers to cross membranes. Physiological buffer systems include: • Phosphate

173

7.13

Biochemistry and Genetics

• Bicarbonate • Protein Of these three systems, proteins are considered the most important because of the large quantity of protein present in the body (about 12 kg). The amino acid histidine is most important because its side chain pKa is not too far from the physiological cellular pH. Also, unlike the phosphate system, the histidine side chain dissociates with a temperature dependence similar to that of water. The buffer systems can be overwhelmed in disease states, leading to acidosis or alkalosis. Types: Respiratory acidosis Retention of carbon dioxide which forms carbonic acid. In lung diseases. Respiratory alkalosis Abnormality low carbon dioxide and carbonic acid because of hyperventilation. Metabolic acidosis Overproduction of organic acids (lactic acid, ketone bodies...), or failure of the kidneys to excrete excess protons. Metabolic alkalosis Abnormal loss of acid, for example by vomiting acidic stomach contents.

7.12. Useful web resources BRENDA enzymological database http://brenda.bc.uni-koeln.de/ IUBMB http://www.chem.qmw.ac.uk/iubmb/ UniProt Universal Protein Resource (sequences), http://www.uniprot.org KEGG Kyoto Encyclopedia of Genes and Genomes (pathway maps), http://www.genome .jp/kegg/pathway.html OMIM online Mendelian inheritance in man (inherited diseases), http://www.ncbi.nlm .nih.gov/sites/entrez?db=omim

174

Example questions

7.13

7.13. Example questions 1) Enzyme turnover rate: An enzyme is turning over at a substrate concentration of [S] = 3 × Km . The reaction velocity will be A) 0.25 × Vmax B) 0.33 × Vmax C) 0.50 × Vmax D) 0.75 × Vmax E) 1.00 × Vmax

2) Enzyme reaction velocity The velocity v

Under conditions of substrate saturation you double [E].

A) remains constant. B) is halved. C) is doubled. D) is increased 10-fold. E) is reduced 100-fold.

3) Enzyme turnover number 5 µg of a pure enzyme (MW = 50 000 Da) give Vmax = 10 µmol/min. What is the turnover number of the enzyme? A) 10 × 103 min−1 B) 25 × 103 min−1 C) 50 × 103 min−1 D) 75 × 103 min−1 E) 100 × 103 min−1

175

7.13

Biochemistry and Genetics

4) Enzyme activity, HMM-equation Two enzymes compete for the same substrate. Most of the substrate will be turned over by the enzyme with A) the highest molecular weight. B) the highest Km value. C) the highest activity and the lowest Km value. D) the lowest activity and highest Km value. E) the highest specificity for the substrate.

5) Action of pharmaceutical You are employed by a drug company developing a new antibiotic against tuberculosis. Testing the interaction of a candidate substance with an enzyme the bacterium needs for division you obtain the diagram above. Which of the following terms best describes the interaction between the potential drug and the target enzyme? antibiotic and enzyme 100 0.4 k (1/s)

remaining enzyme activity (% of original)

0.5

0.3 0.2 0.1 0

10 20 10 5 3 2 1 0.5 0.3

0

5 10 15 antibiotic [mM]

20

mM mM mM mM mM mM mM mM

1 0

5

10

15 time (min)

A) Competitive inhibition. B) Non-competitive inhibition. C) Uncompetitive inhibition. D) Mixed competitive inhibition. E) Inactivation by a suicide substrate.

176

20

25

Example questions

7.13

6) Metabolic and thermodynamic direction The enzyme glucose phosphate isomerase catalyzes the reaction Fru-6P * ) Glu-6P, ∆G = −1.8 kJ/mol. For this reaction to proceed in the physiological direction (toward Fru-6P) one has to A) remove [Glu-6P] in a second reaction B) remove [Fru-6P] in a second reaction C) do nothing, as reactions proceed towards positive ∆G D) increase the [Fru-6P] E) increase the enzyme concentration

7) Henri-Michaelis-Menten law, Lineweaver-Burk plots The diagram shows the LineweaverBurk-plot of an experiment. The interaction is Lineweaver-Burk plot 4

1 / v (s/nmol)

[I] = 0.0 3.5 [I] = 0.5 [I] = 1.0 [I] = 2.0 [I] = 4.0 3 [I] = 6.0 [I] = 10

mM mM mM mM mM mM mM

2.5

2

1.5

1

0.5

0

-2

-1.5

-1

-0.5

0 0.5 1 1 / [S] (1/mM)

1.5

2

2.5

3

A) the competitive inhibition of an enzyme with a Km of 1 mM. B) the competitive inhibition of an enzyme with a Km of 2 mM. C) the non-competitive inhibition of an enzyme with a Km of 1 mM. D) the uncompetitive inhibition of an enzyme with a Km of 1 mM. E) the uncompetitive inhibition of an enzyme with a Km of 2 mM.

177

7.14

Biochemistry and Genetics

8) Hill-equation, co-operative binding Oxygen binding to hemoglobin (in % of maximum) is a sigmoidal function of oxygen concentration (in kPa partial pressure). Assuming a Hill-coefficient h = 2.9 and K0.5 = 13 kPa, what is the saturation of hemoglobin in working muscle, where the oxygen concentration is 1 kPa? A) 0 % B) 7 % C) 20 % D) 50 % E) 100 % 9) difference between inhibition and inactivation Some phosphoesters are used as insecticides (Parathion, E605) and chemical weapons (VX, sarin, tabun). They act by covalently modifying Ser-groups in the active center of acetylcholine esterase in the synaptic gap of the neuro-muscular junction. This reaction most likely results in a A competitive inhibition B uncompetitive inhibition C non-competitive inhibition D mixed inhibition E inactivation 10) Enzyme nomenclature, enzyme classes The reaction depicted is catalyzed by a H2C OH

H2C OH HO CH

+

ATP

C OH H2 Glycerol

A) Oxidoreductase B) Transferase C) Hydrolase D) Lyase E) Isomerase

178

HO CH

O

C O P O H2 O Glycerol-3-phosphate

+

ADP

Objectives

7.14

7.14. Objectives After completion of this course unit students should be able to • list the major classes of enzymes: Oxidoreductases, transferases, ligases, lyases, hydrolases and isomerases and describe the reactions performed by them. • describe the interaction of substrates and products with an enzyme and explain how this leads to catalysis. • list the basic assumptions made in Michaelis-Menten kinetics. • compare the effect of enzymes on thermodynamic and kinetic properties of biochemical reactions. • use the Henri-Michaelis-Menten-equation and the Hill-equation to predict the velocity of enzymatic turnover. • state the importance of Km and Vmax for the rate of enzymatic reactions at high and low substrate concentrations. • explain the role of the efficiency constant Vmax /Km • interpret substrate-velocity and Lineweaver-Burk-plots. • describe the effects of temperature, ionic strength and pH for the rates of enzymatic reactions. • describe the reaction mechanism of competitive, uncompetitive, non-competitive and mixed inhibitions and state how these can be distinguished in a Lineweaver-Burkplot. • describe the difference between inhibitor and inactivator and give examples for the pharmacological use of both. • describe, using examples, some of the mechanisms by which enzymes act. • describe, using examples, the roles of co-substrates and prosthetic groups in metabolism. • describe the structure of ATP, with emphasis on its energy rich bonds and list the reaction types in which ATP participates. • define the energy charge and state the relative abundances of ATP, ADP, and AMP in healthy and metabolically stressed cells. • relate the coenzymes NAD+ , NADP+ , FAD to the reaction types in which they participate.

179

8. Methods in Molecular Medicine 8.1. Restriction Endonucleases The DNA in human chromosomes is too big to be manageable in the test tube. The chromosomes have to be broken down into smaller fragments of some hundred or thousand bp. This is done with restriction endonucleases. These enzymes cleave double-stranded DNA very selectively at palindromic sequences. The palindromes are 4–8 bp long. The longer the recognition sequence of the enzyme, the greater is the average size of the fragments generated. Some restriction endonucleases cleave in the middle of their recognition sequence and produce blunt-ended fragments. But most make staggered cuts, producing fragments with short single-stranded overhangs (“cohesive ends”). Note that the cohesive end of a restriction fragment can anneal (= base-pair) with the end of any other fragment generated by the same restriction endonuclease. You can even link two restriction fragments covalently. Mix the fragments, let them anneal, then add DNA ligase. Restriction endonucleases are bacterial enzymes that are used by the bacteria as a defense against DNA virus. Bacteria protect sensitive sites in their own DNA by methylation. There are lots of restriction endonucleases and a few hundred of them are commercially available.

8.2. Probes One of the main tools in working with DNA, is a probe that recognizes the DNA of interest while ignoring everything else. A probe is a labeled (fluorescent or radioactive) singlestranded nucleic acid that is complementary to the nucleic acid you are looking for. Most important: A cDNA probe represents the exon sequences of a gene. It will identify any restriction fragment that contains expressed sequences of the gene. A cDNA is the reverse transcript of an mRNA.

181

8.3

Biochemistry and Genetics

An oligonucleotide probe is a synthetic DNA that is complementary to a specific DNA sequence. If used on a restriction digest of genomic DNA, it has to be at least 17 or 18 nucleotides long because otherwise the target sequence may be present in multiple sites in the genome. Oligonucleotides of this length can be synthesized at moderately high cost, when 32 P- or fluorescently-labeled. The probe hybridizes with single-stranded DNA (or RNA) that is complementary to its own base sequence. The assay conditions are said to be of high stringency when the probe anneals only with the correct sequence but not with closely related sequences. High temperature, low ionic strength and alkaline pH interfere with annealing and therefore represent conditions of high stringency. Under proper stringency conditions, a single mismatch can prevent the hybridization of an oligonucleotide probe with its target sequence.

8.3. Southern Blotting A restriction digest contains thousands to millions of restriction fragments. Southern blotting combines electrophoretic separation and probing to identify interesting restriction fragments. 1. Treat the DNA with a restriction endonuclease. 2. Separate the restriction fragments by electrophoresis on a cross-linked gel (agarose or polyacrylamide). These methods separate the restriction fragments by size: small fragments move fastest. This gives you a pretty accurate estimate for the size of the restriction fragments. 3. Dip the gel in a NaOH solution to denature the restriction fragments. 4. Transfer (“blot”) the denatured DNA to a nitrocellulose filter. Nitrocellulose binds single-stranded DNA very tightly. Blotting produces a replica of the electrophoretic separation on the nitrocellulose. 5. Incubate the nitrocellulose filter in a solution of the probe and wash off excess probe. 6. Visualize the bound probe by autoradiography or fluorescence scanning. This procedure answers two questions: 1. Is a sequence complementary to the probe in your DNA extract? 2. How long is the restriction fragment detected by the probe?

182

The Polymerase Chain Reaction (PCR)

8.4

Northern blotting is a similar procedure for RNA: the RNA (usually mRNA) is separated by gel electrophoresis, blotted to nitrocellulose, and probed. Restriction enzyme digest and NaOH treatment is not included in Northern blot as opposed to Southern blot. Western blotting is a method for the separation of proteins. The proteins are denatured with the anionic detergent SDS, separated by one- or two-dimensional gel electrophoresis, blotted to nitrocellulose, and “probed” with a (monoclonal) antibody. Eastern blotting works similar, except that the cationic detergent CTAB is used for denaturation, hence polarity during electrophoresis and blotting is reversed.

8.4. The Polymerase Chain Reaction (PCR) PCR is a method for the enzymatic amplification of double-stranded DNA in vitro. This is the second major tool in the DNA laboratory. It can be done on crude DNA extracts, but only a selected segment is amplified. You have to know at least the flanking sequences of the DNA you want to amplify. You need: 1. The extracted DNA. 2. Substrates for DNA synthesis: dATP, dGTP, dCTP, dTTP. 3. A heat-stable DNA polymerase (usually Taq polymerase from the thermophile bacterium Thermus aquaticus). This enzyme polymerizes DNA at 60 °C and survives repeated heating to 95 °C. Like other DNA polymerases, it requires a primer to start DNA synthesis. 4. A pair of oligonucleotide primers that hybridize with complementary sequences on each of the DNA strands. They mark the ends of the amplified sequence. Procedure: 1. The DNA is mixed with deoxyribonucleotides, Taq polymerase, and a very large (>million fold) excess of the primers. 2. The DNA is denatured by heating to 95 °C. 3. The solution is cooled to 60 °C. The primers anneal and Taq polymerase synthesizes new DNA starting from the 3’ ends of the primers. The exact temperature depends on the primer sequences. 4. After 2 or 3 minutes, when the Taq polymerase has worked its way from one primer to (and beyond) the level of the next, the solution is heated again to separate the strands.

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Biochemistry and Genetics

5. The solution is run through 20–30 cycles of heating and cooling. In theory, the amount of DNA between the primers doubles in each cycle. 3' 5' 3'

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(figure from [Buxbaum, in press]) PCR generates a PCR product. This is a blunt-ended double-stranded DNA whose ends are marked by the primers. Indeed, the primers are incorporated in the PCR product. PCR products can be probed, but it is usually more convenient to simply electrophorese the product and stain the gel with ethidium bromide. There is so much more PCR product than other DNA that you can always recognize the product as a band and you are rarely able to see the starting material in the gel. Because of its sensitivity (only a few DNA molecules are needed), PCR is the workhorse for all those applications where only minute amounts of DNA are available, including prenatal diagnosis, preimplantation diagnosis, forensic applications, and the study of fossil DNA. Limitations: • Taq polymerase does not proofread its product, therefore the error rate is high. In part for this reason, the method is suitable only for the amplification of short pieces of DNA, up to 3 kbp. • Because of its high sensitivity, contamination with extraneous DNA is a big problem.

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Gene Mapping

8.6.1

8.5. DNA Sequencing and Deduced Functions Historically, there were two important methods of DNA sequencing, the Maxam-Gilbert Method and the Sanger Dideoxy Method. Today, only the Sanger method is in use. Both methods require a single-stranded DNA, and they determine the sequence of up to several hundred bases in one sitting. We know our chromosomal DNA sequences, but we still need to determine which sequences encode functional gene products, and what those gene products accomplish in the body. Genes can be recognized in newly-sequenced DNA by telltale signs like the TATA box (DNA = TATAAA, start codon (mRNA = AUG), stop codon (mRNA = UAA, UAG, UGA), polyadenylation signal (mRNA = AAUAAA) and splice sites (Intron-exon junctions; Introns begin with RNA = GU and end with AG; there are longer but degenerate consensus sequences for these boundaries). If you find a new gene, you can sometimes deduce its function. • Genes, and the proteins they encode, come in families whose members are structurally related. • If the amino-terminus of the encoded protein is a signal sequence, the protein is most likely a membrane protein or secreted protein. • Hydrophobic sequences of about 20 amino acids in the encoded polypeptide suggest membrane-spanning α-helices. - The presence of specific amino acid sequences may suggest glycosylation or phosphorylation of a gene product.

8.6. Gene Mapping Even with the complete genome sequence in hand, the mapping of genes is a major task in modern genetic research. It is used to map: • Genes for Mendelian disorders • Genes for normal polymorphisms (blood groups, etc.). • “Susceptibility genes” for multifactorial diseases (diabetes, Alzheimer). • Genes affecting continuously variable traits (blood pressure, intelligence, divorce risk...)

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8.6.3

Biochemistry and Genetics

8.6.1. Fluorescent in-situ Hybridization (FISH) A strongly fluorescent cDNA probe is hybridized to a prophase or metaphase chromosome spread. A banding technique is used, and this permits the assignment of the gene to a specific chromosome band. This method can be used whenever a larger genomic probe (> 25 000 bp) is available. Sometimes, a cDNA probe can be used.

8.6.2. Deletion Mapping Most disease-causing mutations are loss-of-function mutations that lead to a nonfunctional or absent gene product. Occasionally, a very large deletion that obliterates the gene itself and a variable amount of neighboring DNA (including, perhaps, some neighboring genes) is the cause of an apparent single-gene disorder. If the deletion is large enough for detection by high-resolution banding, the locus can be assigned to a chromosome band with an accuracy of ± a few million base pairs. In other cases, a single-gene disorder is caused by a chromosomal rearrangement when the breakpoint is within the gene.

8.6.3. Linkage Analysis Genes that are close together on the same chromosome are genetically linked: They segregate together during meiosis. With increased distance of the loci, however, there is an increased chance of crossing-over. A distance on the chromosome with a recombination frequency of 1 % is called one centiMorgan. It corresponds to about 1 million base pairs of DNA. Genes can sometimes be mapped if the locus of an unknown gene is close to that of another gene with known position. Example: The genes for hemophilia and color blindness are about 10 centiMorgans apart on the X-chromosome; and the hemochromatosis gene is closely linked with the genes for the HLA antigens on chromosome 6. The most important genetic markers, however, are not genes but different anonymous DNA markers • A classical restriction fragment length polymorphism (RFLP) is caused by a cleavage site for a restriction endonuclease that is sometimes present and sometimes absent. Two fragments of different length are generated that can be analyzed by Southern blotting. These are rarely used. • A variable number of tandem repeats (VNTR) is a short (2 to some dozen base pairs) sequence that is tandemly repeated many times. Most VNTRs are not in the coding regions of genes but in untranscribed spacers or introns. Good VNTRs are highly polymorphic, with many “alleles” in the population. Ideally, most people in the population should be heterozygous for the VNTR. VNTRs are analyzed by Southern blotting or PCR. These are the markers most often used. “DNA Fingerprinting” methods involve analysis of VNTR analysis.

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Cloning and Genomic Libraries

8.7

Mathematics: The recombination fraction τ is the probability of recombination between two loci (for example a disease gene and an RFLP). It has a numerical value between 0 and 1 and corresponds to the distance in centiMorgans. The likelihood ratio can be calculated for different recombination fractions. It is defined as: The probability that the observed association occurs if there is linkage (with an arbitrarily assumed value of τ ) divided by the probability for this result if there is no linkage. A high likelihood ratio means that the observed results are likely to be caused by linkage rather than the random co-inheritance of two unlinked traits. The lod score is the logarithm of the likelihood ratio. A lod score of >3 is taken as reasonably good evidence for linkage. Formally, it corresponds to a likelihood ratio of 1000; in reality, a better description is that a LOD score of >3 corresponds to the usual p<0.05 limit familiar from statistical analysis. Linkage studies require large families with multiple affected family members. They can be used even if absolutely nothing is known about the structure of the gene and its product, but you need to know the inheritance pattern. If you don’t know on which chromosome the gene is, you may have to make a genome-wide scan, tracking the inheritance of your gene with a few hundred VNTRs scattered all over the 22 autosomes. Linkage studies can only determine the approximate genomic location of a disease gene. The gene itself has to be identified by the study of candidate genes in the relevant area and by DNA sequencing.

8.6.4. Candidate Genes Some diseases give clues about the nature of the gene product. Many connective tissue diseases, for example, are caused by abnormalities of extracellular matrix proteins (collagen, elastin etc). If you have narrowed down the position of a disease gene by linkage studies, you can obtain sequence information for genes in the region from DNA data banks. You can then tentatively identify “suspicious” genes and see if your patients have mutations in these candidate genes. This involves mutation scanning and/or the actual sequencing of the candidate gene in the patients. However, not all mutations cause disease. You may stumble on a normal polymorphism that has nothing to do with the disease!

8.7. Cloning and Genomic Libraries DNA can be amplified by PCR. It can also be amplified by incorporation into a bacterium where it is replicated along with the bacterial DNA. This is called cloning. A clone is a “family” of cells that are descended asexually from a common ancestor. Foreign DNA (for example human genomic DNA) is not incorporated into the bacterial chromosome but into a cloning vector.

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8.7

Biochemistry and Genetics

The cloning vector may be plasmid: a circular, double-stranded DNA that has a replication origin and that carries genes. Most bacteria naturally contain plasmids. Those plasmids that are used as cloning vectors are R-factors that contain at least one gene for antibiotic resistance. Also temperate bacteriophages, like λ phage, can be used as vectors. Many cloning vectors are artificial constructs that were patched up from bits and pieces of plasmids and bacteriophages. The foreign DNA is ligated into the vector DNA in vitro. The recombinant vector is then spirited into the bacterial host cell where it replicates along with the bacterial DNA. Cloning can be used to make a genomic library. This is a large collection of bacteria, each containing a piece of human genomic DNA. In order to be useful, a genomic library has to have every genomic DNA sequence represented in at least one bacterium. Simplified procedure for constructing a genomic library with a plasmid vector: 1. Take an R-factor plasmid that contains a gene for ampicillin (type of weak penicillin) resistance, and cleave its DNA with a restriction endonuclease. You have to use a very selective restriction enzyme that cleaves the plasmid at only one site, outside the resistance gene, creating a linear DNA with single-stranded cohesive ends. 2. Extract human genomic DNA and cleave it with the same restriction enzyme that was used for the plasmid. 3. Mix the cleaved human and plasmid DNA, let them anneal with their sticky ends, then add DNA ligase: human DNA and plasmid DNA become covalently linked into a circle. 4. The engineered plasmid is brought into the bacterium by transformation. This is a low-efficiency process but can be achieved in satisfactory yield in the presence of high calcium concentration. 5. The selection of transformed clones is achieved by plating the bacteria on agar plates in the presence of ampicillin. Only transformed bacteria can survive. Each colony is a clone that contains a plasmid, hopefully with a piece of human DNA. 6. The colony pattern of the agar plate can be transferred to other agar plates. This is called replica plating. 7. The colonies of a replica plate are lysed with NaOH. The denatured DNA is replicaplated to nitrocellulose. After drying, the nitrocellulose filter is dipped into the solution of a probe which identifies a specific sequence of human DNA. This step is called screening. Plasmid vectors are good to propagate small pieces of DNA, up to 5000 bp or 5 kbp. Larger chunks (≈ 15 kbp) can be cloned in λ phage: the “non-essential” phage genes are replaced by an insert (= foreign DNA), and the engineered DNA is packaged into a phage particle in vitro. Only DNA of the right size is packaged. The engineered phage is brought into the cell

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cDNA Cloning and Expression Cloning

8.9

and propagated by lysogenic infection. Very large DNA pieces can be cloned in yeast cells in the form of yeast artificial chromosomes (YACs), patched up from centromere sequences, telomere sequences, and cloned DNA.

8.8. cDNA Cloning and Expression Cloning Only 1–2 % of the DNA in a genomic library is coding. If you are only interested in coding DNA, you better make a cDNA library. A cDNA is a double-stranded DNA copy of a RNA, made by the retroviral enzyme reverse transcriptase. In genome sequencing, cDNAs or fragments of cDNAs are referred to as expressed sequence tags (ESTs). Recipe: 1. Isolate mRNA by affinity chromatography on an oligo-dT column. The poly-dA tail sticks to the immobilized poly-dT. 2. Add oligo-dT primers, reverse transcriptase, and deoxyribonucleotides. The oligo-dT primer hybridizes with the poly-A tail, and the reverse transcriptase synthesizes the cDNA. 3. Ligate the cDNA into a vector, then proceed as usual. The tissue of origin doesn’t matter for a genomic library because all cells have the same genome. But it is important for a cDNA library because different cells express different genes. Ordinarily, neither cloned genomic DNA nor cDNA is expressed in bacteria. For expression cloning, you have to ligate: • A human cDNA. Genomic DNA won’t do because bacteria cannot remove introns. • A bacterial promoter. You need a strong promoter, or one that can be regulated at will. For example, with the promoter of the lac operon, the cloned gene is expressed only when the bacteria are kept on a glucose-free, lactose-rich diet. • The cDNA of a bacterial ribosome-binding sequence (Shine-Delgarno sequence) has to be ligated between the cDNA and the promoter. • A bacterial signal sequence can be included if desired. In this case the bacteria will secrete the protein. With expression cloning, you can turn bacteria into lucrative factories for hormones, clotting factors, cytokines, or any other protein. Only post-translational modifications (phosphorylation, glycosylation) are problematic because bacteria don’t have the right processing enzymes. Many genetically engineered therapeutics are on the market, including human insulin, growth hormone, interferon, clotting factor VIII, erythropoietin and tissue-type plasminogen activator, and humanized antibodies.

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8.10.1

Biochemistry and Genetics

8.9. Site-Directed Mutagenesis and Protein Engineering The base sequence of cloned DNA can be changed intentionally. Cloned DNA is isolated with the help of a restriction endonuclease, modified by enzymatic methods, and re-inserted into a cloning vector. Several methods are available for the introduction of targeted mutations. If you use expression cloning, you can produce proteins with amino acid substitutions or other minimal changes. You can also make truncated proteins that are missing part of the polypeptide, for example soluble variants of the spike proteins of the AIDS virus that interfere with AIDS infection because they jam virus receptors on the cell surface. Domain shuffling is the patching-together of parts from different genes to make a new, recombinant gene (and protein). Example: If you link the DNA-binding domain of the androgen receptor with the retinoic acid-binding domain of the retinoic acid receptor, and engineer this into your tissues, you can improve your virility by gobbling vitamin A.

8.10. Use of DNA Diagnostics The molecular diagnosis of disease genes and normal polymorphisms is the fastest-growing area of modern medical technology. Uses: • Diagnosis of Mendelian disorders • Carrier detection in relatives of patients • Newborn screening • Population-based screening • Predictive testing for late-onset diseases • Susceptibility testing for multifactorial diseases • Prenatal and pre-implantation diagnosis • Paternity testing • DNA fingerprinting of criminal suspects Technical problems for DNA diagnostics include: • Most single-gene disorders show substantial allelic heterogeneity. Therefore, genetic tests must screen for many mutations. • Common diseases are usually multifactorial. Therefore, genetic testing allows only a statistical risk estimate.

190

Southern Blotting with Allele-Specific Probes

8.10.1

8.10.1. Southern Blotting with Allele-Specific Probes Allele-specific probes (ASOs) can be used when both the normal gene sequence and the mutation are known: there is no allelic heterogeneity and no locus heterogeneity. Procedure for sickle cell testing: 1. Make (or buy) two synthetic oligonucleotide probes each of the same length (>18 bp), one complementary to the sequence of the sickle cell mutation and one complementary to the corresponding normal sequence. 2. Treat the patient’s DNA with a restriction endonuclease that cuts left and right of the probed sequence. 3. Divide the restriction digest in two aliquots, and electrophorese them separately in two lanes of a gel. Then blot to nitrocellulose. 4. Apply the probe for the normal sequence to one lane, and the probe for the sickle cell mutation to the other. The stringency has to be adjusted carefully because the two probes differ by only one base. You can distinguish between homozygous normal (only the normal probe binds), homozygous affected (only the sickle cell probe binds) and heterozygous (both probes bind). In this example the restriction fragments have the same length and the same electrophoretic mobility. Example: Adenomatous coli,polyposis an autosomal susceptibility synExample: polyposis Adenomatous coli, andominant autosomalcancer dominant cancer susceptibility drome. Will the children insyndrome. generationWill III the getchildren cancer?in generation III get cancer?

I II

III

Southern Blots Example: Prenatal diagnosis of β-thalassemia. Has the fetus II-3 inherited the disease? Is he a carrier? Is the unaffected daughter II2 a carrier, or homozygous normal?

191

I II

Southern blots

III Southern Blots 8.11

Biochemistry and Genetics

Southern Blots

Example: Prenatal diagnosis of β-thalassemia. Has the fetus II-3 inherited the disease? Is he a carrier? thefetus unaffected daughter II2 a carrier, or Example: Prenatal diagnosis of β-thalassemia. HasIsthe II3 inherited the disease? Is homozygous normal? he a Example: carrier? Is the unaffected daughter II2 a carrier, or homozygous normal? Prenatal diagnosis of β-thalassemia. Has the fetus II-3 inherited the disease? Is he a carrier? Is the unaffected daughter II2 a carrier, or homozygous normal? I

I

II II Southern blots Southern blots

Example: Duchenne muscular dystrophy, a severe X-linked recessive

Example: Duchenne muscular dystrophy, severea X-linked recessiverecessive muscle disease. Are Duchenne muscular severe X-linked Example: muscle disease. Are II2 anddystrophy, II3 acarriers? II2 and II3 carriers? muscle disease. Are II2 and II3 carriers?

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Southern Blots

Southern Blots

77 In the case of small insertions or deletions, only one probe is needed because the restriction 77 fragments differ in length. Allele-specific or organism-specific oligonucleotides are probably more often used in Dot blot analysis thereby saving the time-consuming electrophoresis step.

Limitations: Most single-gene disorders show extensive allelic heterogeneity i.e., many different mutations. Each set of allele specific oligonucleotides will only give you information on one specific mutation. You cannot use allele-specific probes in these cases unless you have identified the mutation in the affected family first, or are prepared to repeat the experiment with many different set of oligonucleotides (and then sequencing would probably be faster and cheaper). Note - A single genetic test sometimes unavailable for genetic disease testing. See the GeneTest website for available tests and clinical testing sites for individual conditions. However, see the section on microarrays.

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Dot-Blotting

8.12

8.11. Use of PCR. PCR requires less DNA than Southern blotting. It is therefore the preferred procedure for prenatal diagnosis and pre-implantation diagnosis. Example: Cystic fibrosis is a severe autosomal recessive disease that is most often caused by a 3-basepair deletion. If both parents carry this mutation (disease risk: 25 %), you can do prenatal diagnosis by Southern blotting. Alternatively, you can use PCR: 1. Amplify a short segment of the fetal DNA (<100 bp) with a primer pair that frames each potential mutation site. 2. Separate the PCR products by gel electrophoresis. Run controls with the normal PCR product and the PCR product with the deletion in separate lanes of the same gel. Then stain with ethidium bromide. If the fetal DNA yields only the normal PCR product, the fetus is homozygous normal; if only the shortened fragment is produced, it is homozygous affected; if both bands are present, it is heterozygous. In the diagnosis of point mutations, the two PCR products have the same length and cannot be distinguished on the gel. Sometimes, a restriction enzyme recognition site is changed by the mutation; then restriction enzyme digest of the PCR product will produce fragments of different lengths. Otherwise, allele-specific probes have to be used. PCR can also be used in cases where we try to detect presence or absence of a given DNA piece. First example in HIV infection: primers specific for the HIV genome will only produce a fragment if the DNA is derived from cells infected with HIV. Second example is diseases caused by deletion of one or more exons in a gene. Primers specific for each exon are used in the PCR, and presence of a fragment will demonstrate presence of the exon in the starting material. Especially in these last applications is there a major problem in securing against accidental contamination with extraneous DNA. The product from last time you performed the procedure can sometime float around in the laboratory in the form of microdroplets.

8.12. Dot-Blotting Dot blotting with DNA samples Both Southern blotting and PCR with electrophoretic separation provide information about the length of a restriction fragment or PCR product. When this information is not needed, and you can use dot-blotting. Procedure: 1. Treat the extracted DNA with a rarely-cutting restriction endonuclease. The probed sites have to remain intact! 2. Denature the DNA, and apply it in single dots to two nitrocellulose filters.

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8.13

Biochemistry and Genetics

3. Probe one filter with a probe for the normal sequence, and the other one with a probe for the mutation. Dot blotting takes far less time than the other procedures. The DNA of many people can be tested simultaneously on the same nitrocellulose filter. Therefore, it can be used for screening programs. Like other allele-specific methods, the usefulness of dot-blotting is limited by genetic heterogeneity. Also, this method tests for only one DNA sequence variant at a time. Dot blotting with RNA samples: The purpose of this procedure is similar to the purpose of Northern blot, i.e., to measure the expression level of a gene in a sample from a tissue. Procedure: 1. Purify total mRNA from the tissues of interest 2. Spot the same amount of mRNA from each sample onto a filter 3. Probe with a cDNA , a single exon, or a long oligonucleotide 4. The resulting signal will be proportional to the concentration of the specific mRNA in the sample Because this procedure omits the gel electrophoresis step of a Northern blot, it is not possible from position information to see that there has been aberrant hybridization to another mRNA, e.g., from a similar gene (you want to check β-hemoglobin; do you get hybridization to α-hemoglobin?). Therefore, the specificity of a probe for Dot blot should be tested in Northern blot before usage in Dot blot. Advantage of Dot-blot for mRNA is speed, larger number of samples on a blot, and fewer steps necessary increasing the chance that the mRNA survives to the time of hybridization (mRNA is a difficult material to work with, RNases are rampant).

8.13. OBJECTIVES IN SUMMARY 1. Outline the procedure of DNA sequencing by the Sanger-Dideoxy and MaxamGilbert methods. 2. Define what is meant by the terms palindromic sequence, cleavage specificity, blunt and sticky ends, cloning site, high/low frequency cutters with respect to restriction endonucleases. Name and describe the function of DNA ligase, reverse transcriptase, polynucleotide kinase, topoisomerase, RNase and DNase. 3. Outline the procedure of Southern blotting and describe the general use of the method.

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OBJECTIVES IN SUMMARY

8.13

4. Outline the procedure of polymerase chain reaction (PCR) and describe the general use of the method. 5. Define what is meant by “restriction fragment length polymorphism” and provide an example of when RFLP analysis would be required to make a clinical decision. 6. Provide a rationale for the use of RFLP analysis and analysis of a VNTR site (Variable Number of Tandem Repeats) in linkage studies. 7. Explain how somatic cell hybrids, in-situ hybridization methods, and linkage studies are useful in gene mapping. 8. Define the recombination fraction and LOD scores. 9. List the steps in cloning foreign DNA into an R-factor plasmid and in λ phage. 10. Compare the procedures for generation of a genomic library, a cDNA library and an expression library; and describe how each library can be screened for a clone of interest. 11. Give examples of proteins produced using genetic technology, and their therapeutic value. 12. Describe procedures for site-directed mutagenesis in cloned DNA. 13. Explain the rationale for using carrier-testing, pre-symptomatic testing, populationbased heterozygote screening, prenatal diagnosis and pre-implantation diagnosis. 14. Specify examples of when Southern blotting with allele-specific oligonucleotide probes is useful to diagnose a known mutation. 15. List the advantages and disadvantages of using PCR-based methods for genetic diagnosis. 16. List the advantages and disadvantages of linkage studies for the diagnosis of genetic diseases. 17. Calculate carrier probabilities and disease risks for Mendelian disorders using linkage studies and closely linked RFLPs. 18. Describe the advantages and limitations of current gene therapy and antisense protocols, including vector design, and clinical rationale. 19. Explain how germline gene manipulation is accomplished, and how knockout and transgenic mice are useful in medical research.

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Part II.

Semester one, Mini II

ROSS UNIVERSITY SCHOOL OF MEDICINE BIOCHEMISTRY AND GENETICS I Handout 11

9. Glycolysis: Splitting glucose in half

GLYCOLYSIS, TCA-CYCLE, OXIDATIVE PHOSPHORYATION

I. Overview 9.1. Overview Glycolysis, TCA cycle and oxidative phosphorylation are the three stages

Glycolysis, TCA cycle and oxidative phosphorylation are the three stages in the catabolism in the catabolism of glucose to CO2 and H2O: of glucose to CO2 and H2 O:

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1. aInmolecule glycolysis, a molecule In glycolysis of glucose (6 carbons)ofis glucose converted (6 to 2carbons) molecules is of converted pyruvate (3 to 2 molecules pyruvate carbons). This pathway ATP (from + carbons).ofThis pathway(3produces 2 ATP (from ADP +produces inorganic 2phosphate) andADP 2 + + + ). Glycolysis takes place in the inorganic phosphate) and 2 NADH (from NAD NADH + H (from NAD ). Glycolysis takes place in the cytoplasm of all cells. cytoplasm of all cells.

Pyruvate 2. entersPyruvate the mitochondria. The mitochondrial converts enters the mitochondria.pyruvate The dehydrogenase mitochondrial pyruvate pyruvate to an acetyl residue (2 carbons) which becomes linked to coenzyme A (CoA). dehydrogenase converts pyruvate to an acetyl residue (2 carbons) which CO2 is released, and one NAD+ is reduced to NADH + H+ for every pyruvate. +

becomes linked to coenzyme A (CoA). CO2 is released, and one NAD is + reduced NADH + Hcycle, for citric everyacid pyruvate. In the TCA to cycle (Krebs cycle), acetyl-CoA reacts with oxaloacetate (4 3. to In form the citrate TCA cycle (Krebs citricreactions acid cycle), acetyl-CoA reacts carbons) (6 carbons). Thecycle, remaining of the cycle regenerate with oxaloacetate (4 carbons) to form citrate (6 carbons). The remaining reactions of the cycle regenerate oxaloacetate from citrate. The TCA cycle forms 2 CO2, 1 GTP, 3 NADH and 1 FADH199 2. 4. The respiratory chain in the inner mitochondrial membrane uses molecular oxygen to re-oxidize the reduced coenzymes, NADH and FADH2. These reactions are coupled to ATP synthesis by oxidative phosphorylation.

9.2.2

Biochemistry and Genetics

oxaloacetate from citrate. The TCA cycle forms 2 CO2 , 1 GTP, 3 NADH + H+ and 1 FADH2 . The respiratory chain in the inner mitochondrial membrane uses molecular oxygen to reoxidize the reduced coenzymes, NADH + H+ and FADH2 . These reactions are coupled to ATP synthesis by oxidative phosphorylation.

9.2. Glycolysis 9.2.1. Reactions of glycolysis The glycolytic reactions are summarized on page 300 of the Meisenberg & Simmons book. Some highlights: • Only the hexokinase, phosphofructokinase, and pyruvate kinase reactions are irreversible under physiological conditions. • Hexokinase is the committed step for glucose metabolism in general, while phosphofructokinase is the committed step for glycolysis. • Two ATP per molecule of glucose are consumed in the hexokinase and phosphofructokinase is the committed step for glycolysis • Two energy-rich intermediates are formed in glycolysis: 1,3-bisphosphoglycerate and phosphoenolpyruvate. These are used for ATP synthesis by substrate-level phosphorylation. Net yield is 2 ATP per molecule of glucose.

9.2.2. Regulation of glycolysis • Regulation reactions are catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase. • In many tissues the enzymes catalyzing the irreversible reactions are induced by insulin and repressed by insulin antagonists (glucagon in the liver). • The most important site for short-term regulation is phosphofructokinase. In the liver, its activity is: – inhibited by ATP and stimulated by AMP (allosterically). – inhibited by citrate (allosterically) - inhibited by low pH. – Stimulated by insulin and inhibited by glucagon.

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Anaerobic glycolysis

9.3

• Other regulated steps: Hexokinase is product-inhibited by glucose 6-phosphate. Pyruvate kinase is allosterically inhibited by ATP.

9.2.3. Inhibition of glycolysis Fluoride ions inhibit enolase. Sodium fluoride is used in the laboratory to inhibit glycolysis in blood samples used for blood glucose determination. Arsenate uncouples substrate-level phosphorylation because it competes with phosphate in the glyceraldehyde 3-phosphate dehydrogenase reaction, forming an unstable product with arsenate instead of phosphate in position 1 of 1,3-bisphosphoglycerate. ‘Uncoupling’ implies that the reactions of the pathway can proceed, but without ATP synthesis. Inherited partial deficiencies of glycolytic enzymes are occasionally seen, most often pyruvate kinase. This causes hemolytic anemia.

9.2.4. Anaerobic glycolysis Glycolysis can produce ATP in the absence of oxygen, but only if NADH + H+ is converted back to NAD+ in the lactate dehydrogenase reaction: NADH + H+ COO

C O CH3

NAD+

-

COO Lactate dehydrogenase

-

HC OH CH3

The LDH reaction is reversible under aerobic conditions but is irreversible under anaerobic conditions when NADH + H+ accumulates. Lactate is a metabolic dead end. It can re-enter the metabolic pathways only via pyruvate. Anaerobic glycolysis, with formation of lactic acid, is the only metabolic pathway that can produce ATP under anaerobic conditions in humans. It is important in: • Cells lacking mitochondria (erythrocytes). • Cells suffering from hypoxia (ischemic tissue). • Exercising muscle Lactic acidosis is the most common form of metabolic acidosis. All conditions in which oxidative metabolism is impaired (pulmonary failure, circulatory collapse, cyanide poisoning...) cause lactic acidosis.

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9.3

Biochemistry and Genetics

9.3. Objectives in Summary 1. State the importance of glycolysis for aerobic metabolism of glucose and the tissues and subcellular location where glycolysis takes place. 2. Know the major reactions and intermediates of glycolysis. 3. Define the term “substrate level phosphorylation”. 4. Write the overall balance of aerobic and anaerobic glycolysis including the number of ATP and NADH + H+ formed. 5. Name the irreversible reactions of glycolysis and the way they are regulated physiologically and the most important situations in which lactic acid accumulates, giving reasons for this accumulation.

202

10. Plasma Proteins 10.1. Plasma Proteins: Overview Plasma is obtained by the centrifugation of whole blood. It is typically 51–55 % of the blood volume in males and 57–59 % in females, the remainder being the packed red cell volume, corresponding to the hematocrit. Plasma contains 6–8 % proteins, including fibrinogen and clotting factors. Serum is obtained by removing fibrin and clotting factors from plasma.

10.1.1. Functions of plasma proteins Colloid-osmotic (oncotic) pressure. Important to counteract pressure filtration of plasma into the interstitial spaces. Necessary to prevent edema. Transport of small metabolites Important for the transport of water-insoluble compounds (cholesterol esters, retinol), and to prevent the renal excretion of valuable watersoluble molecules and ions (hemin, iron, cobalamin). Also transport of many hormones: steroid and thyroid hormones, vitamin D. Defense The immunoglobulins and the components of the complement system are the most notable examples. Blood coagulation. Most of the clotting factors are proteases which eventually convert soluble fibrinogen to insoluble fibrin. Protease inhibitors regulate proteolytic processes, in blood clotting and inflammation. Sources of plasma proteins: Most come from the liver, except immunoglobulins which are made by plasma cells. Half-lives are a few days to a few weeks. Pinocytosis is a non-selective route of removal and degradation. In addition, plasma glycoproteins gradually lose the sialic acid residues from the ends of their oligosaccharide chains while in circulation, and the resulting asialoglycoproteins are taken up into the liver by receptor-mediated endocytosis. With the exception of albumin, almost all plasma proteins are glycoproteins. Chemistry: Molecular weights range from <60 000 to over 700 000. Most have a pI in the acidic range. Acidic pI combined with high molecular weight prevents renal excretion.

203

10.2 Major Plasma Components: Protein Transthyretin

Biochemistry and Genetics

Fraction Prealbumin

Concentration (mg/dL) 10–40

Albumin

Albumin

3500–5000

α1-acid glycoprotein Retinol-binding protein α1-antiprotease Thyroxine-binding globulin

α1 α1 α1 α1

55–140 3–6 200–400 1–2

Transcortin

α1

3–3.5

Ceruloplasmin

α2

15–60

Haptoglobin α2 -macroglobin Hemopexin Fibrinogen C-reactive

α2 α2 β β γ

100–200 150–420 50–100 200–400 <1

Immunoglobulins

γ, also α2 & β

700–1500

Function Binds T4, also with RBP Colloid osmotic pressure, binds many ligands ? Transports retinol Protease inhibitor Binds thyroid hormones Binds glucocorticoids Copper transport? Binds hemoglobin Protease inhibitor Binds heme Clot formation Binding/removal of antigens? Binding and removal of antigens

10.2. Separation of Plasma Proteins Electrophoresis is the most common procedure. The plasma proteins move to the anode at mildly alkaline pH (8.6). After staining, the relative amounts of proteins in the different fractions can be determined by densitometric scanning: 5 fractions are separated: 1. Albumin (55–68 %) 2. α1 -globulins (6–7 %) 3. α2 -globulins (8–9 %) 4. β-globulins (13–14 %) 5. γ-globulins (11–12 %) Only the albumin fraction is reasonably homogenous. All other fractions are mixtures of several proteins.

204

Transport Proteins

10.2.2

Immunoelectrophoresis combines electrophoretic separation with the use of antibodies to identify individual plasma proteins. It is often used to identify the nature of an abnormal protein peak (“paraprotein”) in patients with myeloproliferative disorders.

10.2.1. Albumin Single polypeptide with 585 amino acid residues, 17 disulfide bonds, no carbohydrate. MW 66 000 Da, pI = 4.8. High water-solubility, low viscosity of aqueous solutions. Total amount in the body: 250–350 g. Concentration in plasma: 3.5 g/dL. Also present in interstitial fluid: 40 % of total albumin is in plasma, 60 % in interstitium. Half-life: 17 d. Functions: Osmotic effect: Albumin provides 75–80 % of the colloid-osmotic (oncotic) pressure of the plasma. A decrease of the albumin concentration below 2 % causes edema. Transport: Albumin binds fatty acids, bilirubin, thyroxine, steroid hormones, dicoumarol, penicillin, aspirin, heme, calcium magnesium etc. There are many different binding sites for these ligands. Binding is non-covalent and reversible. Protein binding is important when plasma drug levels are determined in the clinical laboratory: in patients with decreased serum albumin, an increased proportion of the drug is in the free, unbound form. Chemical assays determine the total drug level, but only the unbound form of a drug (or hormone) is biologically active. Analbuminemia is a rare genetic disorder in which albumin is absent or greatly reduced. With abnormalities in lipid metabolism but, surprisingly, little or no edema.

10.2.2. Transport Proteins Transthyretin (prealbumin) migrates faster than albumin in electrophoresis. Binds thyroxine. Binds the RBP-retinol complex and prevents its renal excretion. Retinol binding protein (RBP) transports retinol from the liver to other tissues. Thyroxine binding globulin (TBG) transports thyroxine and T3. 100- fold higher affinity for T4 than prealbumin. Transcortin transports corticosteroids. Sex Hormone Binding Globulin binds androgens and estrogens. Haptoglobin binds hemoglobin. Prevents the renal excretion of hemoglobin after intravascular hemolysis. Hemopexin binds heme and hematin. Prevents their renal excretion. Transferrin transports iron.

205

10.3

Biochemistry and Genetics

The hormone-binding proteins are physiological buffers that regulate the plasma concentration of free (unbound) hormone. Others (transferrin, hemopexin, haptoglobin) bind their ligand and are then removed by endocytosis, followed by utilization or degradation of the ligand and degradation or recycling of the carrier protein. In intravascular hemolysis, oxyhemoglobin dissociates into αβ-dimers which would be lost through the kidneys in the absence of haptoglobin. Also, heme tends to dissociate from the apo-protein, and the free heme, after oxidation to hematin, binds to hemopexin. The hemoglobin-haptoglobin complex is taken up and degraded by reticuloendothelial cells, and the hematin-hemopexin complex by hepatocytes. The levels of haptoglobin and hemopexin are decreased in patients with intravascular hemolysis. This is used to differentiate hemoglobinuria from myoglobinuria in patients with a positive test for “blood” in the urine. The urine test cannot distinguish between hemoglobin and myoglobin, but haptoglobin is low only in hemoglobinuria.

10.2.3. Protease Inhibitors α1 -antiprotease (α1-antitrypsin) is the major component of the α1 -globulin fraction. Also present in external secretions (bronchial mucus). Inhibits a variety of serine proteases. Important to limit proteolysis during inflammation. α1 -antiprotease deficiency is an autosomal recessive trait that affects 1 in 7000 Caucasians (1 in 700 in Sweden). With early-onset lung emphysema, also infantile liver cirrhosis in some patients. Homozygotes (identified by genetic screening) have to avoid smoking. Also, a methionine residue in α1 -antiprotease which is necessary for protease binding is oxidized by cigarette smoke. Laboratory tests for α1 -antiprotease deficiency include plasma protein electrophoresis (decreased α1 -peak) and trypsin inhibitory capacity (TIC) of plasma. α2 -macroglobulin is a major component of the α2 -globulin fraction. Wide range of antiproteolytic activity. No human deficiency states are known.

10.3. Plasma Proteins in Disease States Acute phase reactants increase in response to inflammation or tissue necrosis in infections, trauma, surgery, neoplasms, autoimmune diseases etc. They include α2 -antiprotease, haptoglobin, ceruloplasmin, fibrinogen and C-reactive protein. Characteristic electrophoretic patterns occur in various diseases.

206

Plasma Components for Clinical Use

10.3.1

Electrophoretically observed changes from normal concentrations: Condition Albumin α1 α2 β γ Liver Cirrhosis ↓ ↑↑ Immediate response pattern ↓ ↑ Delayed response pattern ↓ ↑ ↑ Nephrotic syndrome ↓↓ ↑↑ ↓↓ Monoclonal gammopathy ↓ ↑↑↑ Protein malnutrition ↓↓ ↓ ↓ ↓ ↓ Iron deficiency anemia ↑ Albumin concentrations are decreased in many pathological conditions. Only dehydration results in an increase. Relative or even absolute increases in α2 are typical in nephrotic syndrome and proteinlosing enteropathy. In these conditions, plasma are lost in relation to their molecular size: α2 -macroglobulin is retained because it is big (MW 725 000 Da). The α2 -fraction is also increased when haptoglobin (an acute phase reactant) is increased in response to stress. Individual Proteins • Iron-deficiency anemia leads to increased transferrin concentration (increased βglobulin fraction). However, the iron-saturation of transferrin (normal = 30 %) is increased in hemochromatosis and decreased in iron-deficiency. People with very low transferrin levels or congenital atransferrinemia (rare) are susceptible to bacterial sepsis because iron is a limiting factor for bacterial growth in the blood plasma. • Ceruloplasmin is a copper-containing protein of uncertain function. Decreased in Wilson’s disease (hepatolenticular degeneration), an inherited defect of copper excretion into bile. • C-reactive protein (CRP) is the most sensitive acute phase reactant. Levels rise profoundly during infection. Its function is unknown, but it binds to some components of bacterial cell surfaces (including pneumococcal C-protein), activates complement, binds to T-lymphocytes, inhibits clot retraction and platelet aggregation. • Certain proteins not normally present in plasma are useful as markers for specific diseases. α-fetoprotein, which is normally present in fetal plasma but not in adults, is found in most patients with hepatocellular carcinoma.

10.3.1. Plasma Components for Clinical Use Fresh Frozen Plasma is used for hypovolemia, shock, and clotting disorders. It requires 20 min for thawing.

207

10.4

Biochemistry and Genetics

Cryoprecipitate is enriched in factor VIII and fibrinogen. Used for clotting disorders. Available as lyophilized powder. Albumin is available in 5 % and 35 % solutions. For hypovolemia, shock, extensive burns, cerebral edema, protein-losing conditions. Practically no risk of hepatitis transmission. Immune serum globulin is available as 16.5 % solution for i.m. injection. For hypo-gammaglobulinemia, also for prophylaxis of viral hepatitis. Also plasma-free blood cells (RBCs, platelets, granulocytes) are, of course, available to the modern physician.

10.4. Clinical Enzymology Principle: Most of the diagnostically useful serum enzymes are intracellular enzymes that are released into the blood only when the cells get damaged. The enzymes are cleared from the circulation with half-lives of one to several days. A diagnostically useful enzyme should have higher or lower concentration due to a specific disease or condition. Many clinically useful enzymes have isoenzymes forms: Isoenzymes are chemically and biologically distinct forms of an enzyme, often tissue-specific. They catalyze the same reaction but can be distinguished by electrophoresis or differential sensitivity to inhibitors. Mechanism: Tissue enzymes can be released by: Tissue necrosis: Myocardial infarction, hepatitis, acute pancreatitis. Increased membrane permeability: Muscular dystrophy, dermato-myositis, angina pectoris. Increased Tissue Source or Release from Tissue: Neoplasms, psoriasis, Paget’s disease, healing fractures. Impaired Enzyme Excretion: Obstructive jaundice.

Examples: Plasma cholinesterase (“ pseudocholinesterase”) is a normal constituent of plasma. It can degrade certain drugs, including cocaine. Uses: • Cholinesterase is inhibited in organophosphorus poisoning (insecticides, nerve gases). • Activity is depressed in liver diseases (hepatitis, cirrhosis).

208

10.4

Clinical Enzymology

• Cholinesterase activity should be determined in patients to be treated with the muscle relaxant succinylcholine. Cholinesterase normally inactivates the drug, but is deficient in some patients. Alanine transaminase (ALT) and aspartate transaminase (AST) are enzymes of amino acid metabolism that are most abundant in the liver. Aspartate transaminase and to a lesser extent alanine transaminase also occur in other tissues such as skeletal muscle and myocardium. Both enzymes are elevated in liver diseases, AST also after acute myocardial infarction. Alkaline phosphatase (ALP) is increased in 2 types of diseases: Osteoblastic bone diseases. ALP is an osteoblast enzyme that promotes bone mineralization by hydrolyzing pyrophosphate. Plasma levels are increased in all bone diseases with increased osteoblastic activity: healing fractures, osteitis deformans, osteomalacia, rickets, hyperparathyroidism, bone tumors. High levels in growing children and pregnant women during 3rd trimester. Obstructive jaundice The role of alkaline phosphatase in the liver is not known. Besides obstructive jaundice, space-occupying hepatic lesions due to carcinoma, tuberculosis etc. Often result in increased plasma ALP. Isoenzymes: Normal sources of ALP in plasma are bone, liver, intestine, and placenta (3rd trimester of pregnancy). Enzymes from these 4 sources differ in electrophoretic mobility, heat stability, and sensitivity to inhibitors. The clinically important distinction between bone and liver isoenzymes is possible by heating to 56 °C for 15 min. This inactivates the bone enzyme more than the liver enzyme. Acid phosphatase (ACP) occurs in normal prostatic tissue. A different isoenzyme is present in erythrocytes. Used as a marker for metastatic prostatic carcinoma. Not usually elevated in early stages of prostatic cancer. The prostatic enzyme is inhibited by tartrate, the RBC enzyme by copper salts. Lactate dehydrogenase (LDH) is widely distributed, with high levels in myocardium, kidney, liver, muscle. Increased plasma levels are useful in the diagnosis of myocardial infarction, muscular dystrophy, and pulmonary infarction (increased LDH with normal AST 1–2 days after an episode of chest pain), and in monitoring the response to cancer therapy. Isoenzymes: LDH consists of 4 subunits. 2 types of subunit occur, H (in heart) and M (in skeletal muscle), which form 5 isoenzymes. The isoenzymes are numbered according to their electrophoretic mobility: LDH-1 has the greatest anodic mobility, LDH-5 is slowest. Normal plasma contains mostly LDH-4 and -5. Isoenzyme 1 2 3 4 5

Composition H4 H3M H2M2 HM3 H4

Myocardium ++++ ++++ + -

Erythrocytes +++ +++ + -

Skeletal Muscle + ++ ++++

Liver + ++ ++++

Kidney + + ++ ++ ++

209

10.5

Biochemistry and Genetics

After acute myocardial infarction, LDH-1 is higher than LDH-2: flipped LDH. Creatine kinase (CK) is present in high concentration in skeletal muscle and myocardium, also in brain. Very little in other organs, none in the liver. It is a dimeric enzyme, formed from M subunits (skeletal muscle) and/or B subunits (brain). CK-1: BB, in brain CK-2: MB, in myocardium CK-3: MM, in skeletal muscle, also myocardium CK-1 moves fastest during electrophoresis. CK in normal plasma is almost exclusively CK-3. CK-3 is elevated in muscular dystrophy, after surgery, and after i.m. injections. Elevations of CK-2 are diagnostic for myocardial infarction. Elevations of serum CK-1 are rarely observed, even after cerebrovascular accidents. α-Amylase and lipase leak from the pancreas into the bloodstream in acute pancreatitis. Also elevated in patients with bowel infarction or perforation. Acute myocardial infarction leads to elevations of CK, LDH and AST. Typical time course: 5 Elevation above normal (1) 4

CK

(Ratio of 3 Activities) 2

AST LDH

1

Normal Limit 1

2

3

4

5

6

7

8

Days after chest pain episode

The “flipped LDHcan often be observed during the first few days after MI only. If CK remains elevated beyond day 2, it is usually only the CK-3 isoenzyme.

10.5. Objectives in summary 1. Describe the normal pattern of plasma proteins obtained by electrophoresis. 2. Name the binding specificities of serum albumin, transthyretrin, retinol binding protein, the hormone binding proteins and haptoglobin and hemopexin. 3. Describe the clinical consequences of α1 -antitrypsin deficiencies. 4. Name the acute phase reactants and the changes of plasma proteins in infection.

210

Objectives in summary

10.5

5. Describe the plasma protein abnormalities as seen in electrophoresis, in patients with acute diseases, chronic diseases, protein-losing conditions and monoclonal gammopathies. 6. List the therapeutic uses of fresh frozen plasma, cryoprecipitate and albumin. 7. Name diseases for which the determination of serum enzymes is important, including plasma cholinesterase, the transaminases, alkaline and acid phosphatases, lactate dehydrogenase, γ-glutamyltransferase and amylase. 8. State the time frame for the elevation of CK, LDH and AST after acute myocardial infarction.

211

11. Blood Coagulation 11.1. The Biochemistry of Blood Coagulation Thrombosis is the term used to describe clotting of blood. This process involves the formation of a platelet plug at the site of injury, and deposition of a network of fibrin protein. Alternatively, thrombosis which occurs internally can lead to disease states. Patients with inherited deficiencies of clotting factors or acquired deficiencies by environmental effects have poor clinical indications of blood coagulation. Thrombosis is also the cause of many age-related illnesses. Clotting is activated with increasing age when coagulation factors levels and blood vessel weakness present an increased clotting risk. The hematologist needs a basic science understanding of blood clotting as well as a focused approach to family medical history, lab test data, and knowledge of how to extend these analysis by further measurement of specific proteins and DNA sequences when required.

11.1.1. Primary hemostasis (platelet-plug formation) The platelets are highly structured anucleate cell bodies in blood at a concentration of 1.5–4 × 105 µl−1 . Primary hemostasis is triggered by a variety of signals from damaged or activated vascular endothelial cells and/or exposure of the underlying subendothelial matrix. Platelets first adhere to the signaling site via adhesive ligands. The main interaction is between platelet membrane receptor glycoprotein Ib-IX and the giant polymer von Willebrand factor (vWF). Adherent platelets flatten and activate membrane fibrinogen receptors (glycoprotein IIbIIIa) that bind plasma fibrinogen (Fb). The aggregated platelets are activated by turning inside-out and exposing negatively charged phospholipids (esp. phosphatidyl serine and phosphatidyl inositol).

11.1.2. Secondary hemostasis (fibrin clot formation) Blood shear will disintegrate platelet plugs unless a fibrin net forms. The fibrin net is produced by a complex sequence of enzymatic activities catalyzed by activated forms of the factors named below. These reactions constitute a proteolytic cascade, in which one

213

11.1.4

Biochemistry and Genetics

enzymatic reaction catalyzes the activation of the following reaction in a chain - leading to fibrin formation. Tissue Factor (TF) is exposed on activated endothelial cells and leukocytes where tissue is damaged. This protein activates Factor VII. The TF/VIIa complex binds and activates Factor X. The resulting activated Factor Xa moves to the platelet surface. [Note that little ’a’ indicates a clotting factor protein which has been activated into an enzyme of an enzyme cofactor.] If Factor Xa cleaves enough thrombin from the prothrombin precursor, then coagulation will proceed. Coagulation occurs only after thrombin stimulates activation of enough Factors VIIIa, Va and IXa. Factors VIIIa and IXa make the ’tenase’ complex which produces a constant source of Factor Xa. This Factor Xa is located on the platelet surface and interacts with Factor Va to form prothrombinase complexes - rapid activation of prothrombin to thrombin occurs by this activated complex. Thrombin cleaves fibrinogen to form a fibrin clot, and also binds to form a structural component of the protein matrix. Clot Regulation Thrombin binding to thrombomodulin on vascular endothelial cells interrupts the function of Factors Va and VIIIa, thus slowing thrombin formation. The action of the Protein C and S on Factors Va and VIIIa brings coagulation to a halt. By a separate mechanism, the protein antithrombin inactivates fluid-phase thrombin, and other serine proteases, when it is bound to heparin-like proteoglycans. Fibrinolysis Plasmin is an important degrading enzyme, specifically of fibrin clots. It is a serine protease that is released as plasminogen into the circulation and activated by tissue plasminogen activator (tPA), thrombin, fibrin and factor XII (Hageman factor). It is inactivated by a2-antiplasmin, a serine protease inhibitor. Plasmin cleaves the fibrin polymer releasing fibrin dipeptides thus removing the clot.

11.1.3. Clinical Correlates Patient presentation A history is focused on whether the patient’s condition is consistent with excessive bleeding. Some common presenting conditions of patients are hematuria, facile bruising, or bleeding in connection with dental surgery, circumcision, tonsillectomy, nose-bleeds, and rectal bleeding. Note that patient estimates of blood loss by visual estimations are often overestimates. A clinical examination should include careful inspection of the skin, oral mucosa, musculoskeletal system, nervous system, and sites of active blood loss. Venous blood extraction should be performed by an expert, due to the possibility of massive compartment bleeding in patients with bleeding disorders.

214

Features of Coagulation

11.1.5

11.1.4. Laboratory Tests Platelet counts: Anticoagulated venous blood is counted using an automated cytometer counting particles of 2–37 µm−3 . Reduced counts are associated with prolonged bleeding times. Coagulation tests: Prothrombin time (PT): The PT tests the function of the ‘extrinsic’ and B. Laboratory Tests final common clotting pathways. Tissue Factor is added to citrated test plasma 1. Platelet counts:at 37 °C, and the mixture is recalcified. Clot formation occurs in 12– a. Anticoagulated venous blood is counted using an automated 15 s normally. Prolonged times indicate deficiencies. The test result depends on cytometer counting particles of 2-37 μM3. Reduced counts are associated prolonged bleeding times. adequatewithconcentrations of coagulation factors VII, X, V, II and fibrinogen in test plasma. 2. Coagulation tests: a. Prothrombin time (PT): The PT tests the function of the 'extrinsic' and

Activated partial thromboplastin The APTT tests the function of final common clotting pathways. Tissue Factortime is added(APTT): to citrated test plasma at 37 C, and the mixture is recalcified. Clot formation the ’intrinsic’ and final common clotting pathways. Phosphatidyl serine (PS) is occurs in 12-15s normally. Prolonged times indicate deficiencies. The test result on adequateat concentrations of coagulation added intodepends test plasma 37 °C. Contact activation is prevented by addition of factors VII, X, V, II and fibrinogen in test plasma. b. Activated partial thromboplastin time (APTT): The APTT tests the a strong activator (eg. kaolin). Clot formation occurs in 30–40 s. function of the 'intrinsic' and final common clotting pathways. Phosphatidyl serine (PS) is added into test plasma at 37 C. Contact activation is prevented by addition of a strong activator (eg. kaolin). Clot formation occurs in 30 - 40s. c. Thrombin time (TT): Thrombin is added to a test plasma and times to the clot end-point are measured. Abnormal TT (> 15s) are due to low fibrinogen, or inhibitors (eg. heparin), or an abnormal fibrinogen molecule (dysfibrinogenemia).

Thrombin time (TT): Thrombin is added to a test plasma and times to the clot endpoint are measured. Abnormal TT (> 15 s) are due to low fibrinogen, or inhibitors (eg. heparin), or an abnormal fibrinogen molecule (dysfibrinogenemia). VII

TF

X IX V VIII XI XII

Fibrinogen

II

IIa

Fibrin

APTT PT TT 141

These tests are best used together for differential diagnosis of specific clotting defects.

11.1.5. Features of Coagulation Hepatocellular failure has combined effects on coagulation due to: • Reduced plasma coagulation factors

215

11.1.6

Biochemistry and Genetics

• Induction of a hyperfibrinolytic state, further lowering Factor concentrations in blood. • Splenic pooling of platelets due to portal hypertension. Heparin is known to block the function of several clotting factors. This can be a confounding factor because some medical devices are flushed with heparin prior to blood collection. Hemostatic variables depend on age-specific reference ranges, particularly in children and infants (who have different vitamin K levels than adults) and pregnancy. Many clotting factors (II, VII, IX, X) require liver Vitamin K for their biosynthesis. The membrane and calcium associations of these enzymes depend on post-translational modification of γ-carboxylation of glutamic acids. The drug dicoumarol (warfarin) interferes with blood clotting by inhibiting Vitamin K-dependent biosynthesis of the clotting factors. Common clinical vitamin K deficiency occurs in newborns, liver disease, and malabsorption.

11.1.6. Genetics of Blood Coagulation Hemophilia A is an X-linked bleeding disorder resulting from deficiency of Factor VIII. Affected individuals suffer easy bruising, prolonged bleeding from wounds and muscle hemorrhage. Weight bearing joints suffer intracapsular bleeding leading to swelling and inflammation, and eventually amputation. About half of mutations leading to severe hemophilia A arise from inversions in the region Xq28. Milder forms usually arise from missense mutations. Treatment is by recombinant Factor VIII infusions which greatly improves life expectancy. Hemophilia B (Christmas Disease) is an X-linked disorder caused by deficiency of vitamin K-dependent procoagulant protein Factor IX. Clinically indistinguishable from the more common Hemophilia A. Hemophilia most often presents during early childhood. Patients with a long APTT and a normal PT should be tested for Factor VIII and Factor IX levels to differentiate hemophilias A and B. von Willebrand Disease von Willebrand Factor (vWF) is a complex multimeric glycoprotein associated with subendothelial connective tissue. The protein functions to form a bridge between platelets in the area of vascular damage. It also has a role in stabilizing and increasing plasma concentration of Factor VIII. Hence, vWF patients can present with symptoms resembling those of hemophilia or platelet dysfunction disorders. However, the mode of inheritance is autosomal recessive and bleeding is usually observed in mucocutaneous sites rather than joints. This common disorder may prolong the APTT. Immunoassays for vWF protein are used to identify the presence of vWF. This assay may therefore give normal results

216

Objectives in Brief

11.2

in mutations resulting in loss-of-function, with normal or slightly reduced amounts of vWF protein. Treatment with Factor VIII infusions corrects the symptoms of many patients. Purified vWF is also effective, but is more difficult to obtain due to the very large size of the protein. Factor XIII deficiency This enzyme deficiency is due to mutation in one of the two subunits of a transglutaminase enzyme. This enzyme cross-links fibrin polymer by formation of covalent bonds between glutamine and lysine side-chains. This is a rare autosomal recessive disorder. Presentation is in bleeding from the umbilical cord stump in neonates, intracranial hemorrhage, or after surgical challenge in children (often dental extractions). Maintaining levels of Factor XIII at 10–20 % of normal values is sufficient to correct the bleeding disorder. Factor XIII deficiency is often grouped with the dysfibrinogenemias (reduced functional fibrinogen), which result in similar symptoms.

11.2. Objectives in Brief 1. Diagram the blood coagulation process identifying the role of the major participating proteins and enzymes. Platelets, glycoprotein Ib-IX, von Willebrand factor (vWF), fibrinogen, glycoprotein IIb-IIIa, tissue factor (TF), Factor VII, Factor X, thrombin, Factor VIII, Factor V, Factor IX. 2. Explain the mechanisms of clot regulation and fibrinolysis. 3. Name the most common clinical tests performed for coagulation function and comment on the interpretation of their result. 4. Discuss the relation of clinical tests to differential diagnosis of blood clotting disorders. 5. Name the most important genetic conditions which manifest as bleeding disorders. Hemophilia A, Hemophilia B, von Willebrand Disease, and Factor XIII deficiency. 6. Describe the relation between disease symptoms in these inherited disorders and the normal function of the deficient enzyme.

217

12. Blood 12.1. Blood Groups Blood groups are genetic polymorphisms of red blood cells antigens. They are important in blood transfusions, and some can cause maternal-fetal incompatibility.

12.1.1. The ABO system This system contains two effective antigens, A and B. People with neither A nor B have the H-substance (= O-antigen) instead which does not induce antibody formation in humans. The 4 possible phenotypes (A, B, AB and O) are determined by 3 alleles of a single locus, A, B, and O. A and B are co-dominant, O is recessive. Surprisingly, everyone who does not have the A or B antigen on his own cells has a circulating IgM antibody to the missing antigen, even if he has never been exposed to incompatible blood before. Most likely, these antibodies are induced by contact with microbial cell surface carbohydrates shortly after birth. Antigen-antibody reaction results in agglutination of RBCs. Blood group Genotype Antibodies in Blood aggluti(antigens on Serum nates when mixed RBCs) with RBCs O OO anti-A, anti-B A AA or AO anti-B anti-A B BB or BO anti-A anti-B AB AB none anti-A, anti-B In blood transfusions, the donor’s RBCs may be agglutinated by the recipient’s antibodies. Donor Recipient O A B AB O + + + A - + + B + + AB Persons with blood group O are universal donors, those with blood group AB universal recipients. Except in emergencies, however, patients are treated only with blood of their own

219

12.1.2

Biochemistry and Genetics

group to avoid any agglutination of recipient cells by donor serum. Increasingly, transfusion with resuspended packed RBCs is done to avoid this minor type of transfusion reaction. O and A are most common in Europe, AB is least common. The ABO blood group substances are oligosaccharides which occur as components of sphingolipids and glycoproteins. Their antigenic specificity is determined by the terminal sugars. The products of the A and B alleles, which differ by 4 amino acid substitutions, are glycosyltransferases which transfer N-acetylgalactosamine and galactose, respectively, to the end of the oligosaccharide. The product of the O allele, which contains a nonsense mutation, is non-functional. The ABO blood group substances are present not only on RBCs, but on other cells as well. Therefore, ABO matching is essential not only for blood transfusions but also for organ transplantations. In addition, many people secrete ABO antigens, in glycoprotein form, into saliva and other external secretions. This requires the secretor gene Se. The secretor phenotype is determined by the SeSe or Sese genotype, non-secretors have sese. 23 % of Europeans are non-secretors (sese).

12.1.2. The Rhesus System The antigenic determinants in this system are sequence variants in three membrane proteins encoded by two closely related genes. There are multiple alleles, some of them determine rhesus positive (Rh+) and some rhesus negative (Rh-) phenotype. Rh+ is dominant over Rh-. The frequency of the Rh- phenotype is about 15 % in Europe, less elsewhere. Rhesusnegative people do not normally have an antibody to the rhesus antigen although the transfusion of Rh+ blood can induce the formation of an IgG antibody. Rhesus incompatibility between mother and fetus can cause Hemolytic disease of the newborn: A Rh- mother can become immunized to the Rh antigen of her Rh+ fetus. This can occur during birth or abortion. In a next pregnancy with a Rh+ child, the anti-Rh antibody crosses the placental barrier and coats fetal RBCs which are subsequently destroyed. This results in anemia and a compensatory increase of erythropoiesis. Immature RBCs are released into the fetal circulation (“erythroblastosis fetalis”). Severe cases lead to intrauterine edema (“hydrops fetalis’) and fetal death. Hyperbilirubinemia may result in kernicterus. ABO-incompatibility prevents sensitization to the rhesus antigen. Fetal blood cells entering the maternal circulation are destroyed by maternal anti-A or anti-B before they can induce the formation of the Rh-antibody. ABO incompatibility can also cause hemolytic disease of the newborn, but this is very mild because ABO antibodies are IgM while rhesus antibodies are IgG. Only the rhesus antibodies can easily cross the placenta and cause severe hemolysis.

220

Chemical Inactivation of Hemoglobin

12.2.1

Prevention: Passive immunization of the Rh- mother against the Rh antigen at the time of birth prevents sensitization (RhoGAM).

12.1.3. Other blood group systems There are more than 20 other blood group systems. Most of them are important only in blood banking, especially for patients with chronic blood diseases (thalassaemia, hemophilia) receiving repeated blood transfusions. The time-honored use of blood groups for paternity testing is being replaced by DNA tests.

12.2. Structure of Hemoglobin 4 polypeptides (= subunits), each with its own heme: α2 β2 Major adult hemoglobin (HbA) (98 % in adults) α2 δ2 Minor adult hemoglobin (HbA2 ) (2 % in adults) α2 γ2 Fetal hemoglobin (HbF) (two types, both present in the fetus) β, γ and δ-chains (146 amino acids) have very similar primary structures. Structural homologies exist between α-chains (141 amino acids), β-chains and myoglobin, indicating a common evolutionary origin. The tertiary structures of these 3 polypeptides are very similar. Hemoglobin is similar to 4 myoglobin molecules in structure. The subunits interact by salt bonds and hydrogen bonds. Heme binds to the apo-protein as in myoglobin. Each hemoglobin molecule binds 4 O2 -molecules.

12.2.1. Chemical Inactivation of Hemoglobin Methemoglobin is hemoglobin in which the Fe2+ (ferrous) is oxidized to Fe3+ (ferric). It cannot bind oxygen. Causes for excessive methemoglobin formation: • exposure to oxidizing chemicals (most common cause) • deficiency of methemoglobin reductase in erythrocytes, an enzyme which reduces methemoglobin back to normal hemoglobin • structural abnormalities of hemoglobin affecting the binding of heme to the apoprotein.

221

12.2.2

Biochemistry and Genetics

Methemoglobinemia is treated by the administration of a reducing agent (methylene blue). Carbon Monoxide (CO) binds to the heme iron of hemoglobin and myoglobin as oxygen, but with a 200-fold higher affinity: it prevents oxygen release from the three remaining sites and impairs oxygen delivery. CO is a product of incomplete combustion in automobile exhaust gas, cigarette smoke, and burning buildings. A very small amount is formed endogenously. CO-poisoning is treated with hyperbaric oxygen. Color: Oxygenated hemoglobin: red Deoxy-hemoglobin: blue CO-hemoglobin: cherry-red Importance of apo-protein-heme association: • The apo-protein protects heme from oxidation to hemin (hemin = heme containing Fe3+ ). • CO-affinity is reduced by the distal histidine. Heme in solution (without apo-protein) has a 25 000 times higher affinity for CO than for O2 .

12.2.2. Allosteric Properties

Allosteric proteins can assume alternative conformations. They usually consist of more than one subunit. In hemoglobin, the conformation that prevails in oxyhemoglobin is called the R-form (R = relaxed), the conformation of deoxy-hemoglobin is called the T-form (T = tense). O2 binding breaks the salt bonds between the subunits and rearranges the intersubunit hydrogen bonds, thereby changing the quaternary structure from T to R. The R-form has a 300 fold higher O2 -affinity than the T-form. Therefore there is positive cooperativity between O2 -binding sites: binding of O2 to one heme increases the O2 -affinity of the other 3 hemes. This positive cooperatively results in a sigmoidal O2 -binding curve:

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12.2.2

Allosteric Properties

venous pO2 working

arterial pO2 resting

100

Oxygen Saturation (%)

80

60

haemoglobin myoglobin w/o cooperativity

40

20

0 0

3

6

9

12

15

pO2 (kPa)

Myoglobin has no allosteric properties and no positive cooperativity (hyperbolic O2 -binding curve). The P50 is the O2 partial pressure resulting in 50 % saturation of the oxygen carrier. The P50 of hemoglobin is 3.5 kPa; and of myoglobin 0.07 kPa. A ligand is any substance that binds to the protein in question: O2 is the most important ligand for hemoglobin. An allosteric effector is a “regulatory ligand” that influences the equilibrium between the alternative conformations of an allosteric protein. It binds to a site distinct from the binding site for the functional ligand (O2 in the case of hemoglobin). A positive allosteric effector favors the ligand-binding form, and a negative allosteric effector favors the non-ligandbinding form. 2,3-Bisphosphoglycerate (BPG) is a negative allosteric effector of hemoglobin. In RBCs, it is present in roughly equimolar concentration with hemoglobin. It binds to the β-chains of hemoglobin in the T-conformation but not the R-conformation, thereby stabilizing the T-conformation and lowering the O2 -affinity. It is in large part responsible for the lower O2 -affinity of hemoglobin as compared to myoglobin. Its concentration increases during adaptation to oxygen-deficient conditions (high altitude).

223

12.2.2

Biochemistry and Genetics Oxygen binding to HbA: Effect of 2,3-BPG

100

Oxygen Saturation (%)

80

60

40

0.0 0.5 1.0 2.0 6.0

mM mM mM mM mM

20

0

0

1

2

3 pO2 (kPa)

4

5

6

Homotropic effects in allosteric proteins are interactions between identical ligands: positive cooperativity of O2 -binding in hemoglobin. Heterotropic effects are interactions between different ligands: O2 and BPG in hemoglobin. The Bohr-effect is the reduction of the O2 -affinity by increased acidity and [CO2 ] (in exercising muscle). Bohr-effect 100

Oxygen Saturation (%)

80

60

40

pH 7.75 pH 7.50 pH 7.25 pH 7.00

20

0 0

1

2

3 pO2 (kPa)

4

5

6

Acid increases the [H+] lowering Hb oxygen-affinity because oxyhemoglobin is more acidic than deoxy-hemoglobin:

224

The Hemoglobinopathies

12.2.3

Hb + O2 * ) Hb:O2 + n H+ Increased [H+ ] shifts the equilibrium of this reaction to the left. CO2 reduces the oxygen-affinity through its reversible, nonenzymatic, covalent binding to the terminal amino groups to form carbamino-hemoglobin. R-NH2 + CO2 * ) R-NH-COO− + H+ In carbamino-hemoglobin, the T-conformation is favored and the O2 -affinity decreased. Fetal hemoglobin (α2 γ2 ) has a higher O2 -affinity than HbA because BPG is less tightly bound. But its O2 -affinity is not as high as that of myoglobin. Myoglobin does not respond to BPG, H+ or CO2 . Most of the carbon dioxide is transported as HCO− 3 , formed from CO2 + H2 O by carbonic anhydrase in RBCs. Smaller amounts are transported as dissolved CO2 and as carbaminohemoglobin.

12.2.3. The Hemoglobinopathies Globin Genes There are 2 clusters of globin genes: α-like genes (chromosome 16) 2 identical α-chain genes, ζ-chain gene. β-like genes (chromosome 11) β, δ,A γ,G γ genes, A γ and G γ code for two γ-chains that differ in only one amino acid (Ala versus Gly). Both clusters contain nonfunctional pseudo-genes as well. Expression: ζ and  are expressed only in the embryo, α and γ in the fetus (2nd and 3rd trimester), β and some δ in adults. At birth, 75 % of hemoglobin is HbF (α2 γ2 ) and 25 % is HbA (α2 β2 ). HbF disappears within 4–6 months after birth. Hemoglobin Point Mutations More than 400 single-amino acid substitutions in α- or β-chains are known. Consequences: • More than half of these mutations are asymptomatic. • Mutations in the heme-binding pocket are likely to cause methemoglobin. • Some mutant hemoglobins have an abnormally low oxygen affinity (causing cyanosis) or an abnormally high oxygen affinity (causing polycythemia).

225

12.2.3

Biochemistry and Genetics

• Some abnormal hemoglobins have reduced solubility, leading to sickling.

Sickle Cell Disease Mutation: A point mutation leading to a Glu→Val Substitution in position 6 of the β-chain (HbS). Properties: Normal O2 - affinity, but reduced solubility of deoxy-HbS, with sickling of RBCs in venous (but not arterial) blood. Inheritance: A/S heterozygotes are healthy although their RBCs can sickle under oxygenfree conditions in the test-tube. S/S homozygotes have sickle cell disease. Signs & Symptoms:

• No problems up to 4–8 months

• Moderately severe anemia (total hemoglobin 7–11 %). • Acute “sickling crisis”: Painful crisis (most common), splenic sequestration crisis (infants/young children only), aplastic crisis. • Multiple infarction: “Autosplenectomy”, renal infarcts, leg ulcers, cerebrovascular accidents. • Increased mortality: Infections and sequestration crisis in children, cerebrovascular accidents and renal failure in adults. Diagnosis: The blood smear shows irreversibly sickled cells; HbS solutions become turbid in an oxygen-free environment; hemoglobin electrophoresis; DNA probes. Treatment: Avoidance of hypoxia, fluids and analgesics during acute attack, symptomatic treatment of complications, drugs that increase HbS-solubility or induce HbF synthesis. Prevalence: High in Africa, Arabia, India, and Mediterranean. Heterozygotes are partially protected from malaria.

Hemoglobin C: Glu → Lys substitution in position 6 of the β-chain. Mild hemolysis in C/C homozygotes, mild form of sickle cell disease in S/C heterozygotes.

226

The Hemoglobinopathies

12.3.1

Thalassemias The synthesis of either α-chains or β-chains is reduced or absent, leading to anemia of varying severity. Common in the Mediterranean, Africa, South and Southeast Asia. α-thalassaemia: Not enough α-chains β-thalassaemia: Not enough β-chains Thalassaemia minor: Heterozygous thalassemia. With borderline anemia. Thalassaemia major: Homozygous thalassemia. Severe anemia.

α-Thalassemia Usually caused by large deletions of α-chain genes. 1 gene deleted: Silent carrier. Asymptomatic 2 genes deleted: α-thalassemia minor. Borderline. 3 genes deleted: Hemoglobin H disease (HbH = β4 ). Moderately severe anemia. 4 genes deleted: Hemoglobin Barth disease (Hb Barth = γ4 ), hydrops fetalis.

β-Thalassemia More than 90 different mutations are known. Heterozygotes are borderline normal/anemic. Cooley’s anemia (= β-thalassemia major): Either with complete lack of β-chains (β0 thalassemia) or a very low amount of β-chains (β+ -thalassemia major). Milder variants are called β-thalassemia intermedia. Most “homozygotes” are actually compound heterozygotes. Signs & Symptoms: β0 -thalassemia with severe anemia (2–5 % hemoglobin). Only HbA2 and HbF. Microcytosis, poikilocytosis, anisocytosis, target cells. Abortive erythropoiesis, abnormal expansion of red bone marrow, extramedullary erythropoiesis (in liver). Untreated patients die in childhood. Treatment: Regular blood transfusions. Chronic infusion of desferrioxamine (iron chelator) to prevent iron overload. Bone marrow transplantation, drugs inducing γ-chain (HbF) expression. Bone marrow transplant.

227

12.3.2

Biochemistry and Genetics

12.3. Objectives in Summary 12.3.1. Blood Groups 1. Describe the biochemical background for ABO and Rhesus blood groups. 2. Use the ABO, rhesus and MN blood group systems for paternity testing. 3. Predict transfusion reactions due to ABO incompatibility for all possible combinations of donor and recipient. 4. Outline the pathogenic mechanism of hemolytic disease of the newborn due to rhesus incompatibility or to ABO incompatibility; compare and contrast these two.. 5. Describe the mechanism by which ABO incompatibility can protect from hemolytic disease due to rhesus incompatibility, and give the rationale for the use of RhoGAM.

12.3.2. Hemoglobin and Myoglobin Biochemistry 1. Describe the structure of myoglobin with respect to α-helical structure, the role of nonpolar interactions for the tertiary structure, and the roles of non-covalent interactions, proximal histidine and distal histidine for heme binding to the apo-protein. 2. State the reversible, non-covalent nature of oxygen binding to the heme iron and the importance of the iron being in the ferrous state. 3. Define the terms hematocrit, anemia, polycythemia, and hemolysis. 4. State the typical normal value for hematocrit and blood hemoglobin concentration. 5. Describe the subunit structure of hemoglobins A, Az, and F and specify the interactions between the subunits. 6. State the chemical difference between hemoglobin and methemoglobin and name causes of methemoglobin formation. 7. Describe the competitive interaction between carbon monoxide and oxygen in carbon monoxide poisoning and name the therapeutic approach. 8. Define the term “allosteric protein”, “positive allosteric effector”, “negative allosteric effector”, “homotropic effect” and “heterotropic effect”. 9. Describe the oxygen binding curves for myoglobin, adult hemoglobin and fetal hemoglobin, and relate the sigmoidal binding curves of the hemoglobins to their allosteric properties.

228

Hemoglobinopathies

12.3.3

10. Describe the binding of 2,3-bisphosphoglycerate to hemoglobin. Describe the effects of acidity and carbon dioxide concentration on oxygen binding in the Bohr effect.

12.3.3. Hemoglobinopathies 1. Describe the molecular defect in sickle-cell disease and how this leads to its clinical expression. 2. Define the categories of thalassemias in terms of their associated molecular lesions and severity. Explain how the thalassemias present, clinically. 3. List the treatment options for the major hemoglobinopathies.

229

Part III.

Semester one, Mini III

13. Mendelian Inheritance 13.1. The Patterns of Mendelian Inheritance Definitions: Alleles: Alternative forms (variants) of a gene. Locus: The position of the gene on the chromosome. Homozygous: Carrying 2 identical alleles of a gene. Genotype: The genetic constitution. Usually used with reference to a specified gene. Phenotype: The appearance of the individual. Wild-type allele: The “normal” variant of a gene. Compound heterozygote: Person carrying two different mutations in the two copies of a gene. Dominant allele: Allele that determines the phenotype both in the homozygous and the heterozygous state. Recessive allele: Allele that determines the phenotype in the homozygous but not the heterozygous state. Co-dominant alleles: Both phenotypes are partially expressed in the heterozygous state. Proband (propositus): The individual who brings the family to the doctor’s attention. The phenotype can be analyzed at various levels. In many metabolic disorders the heterozygotes are healthy (recessive inheritance), although the activity of the affected enzyme is intermediate (co-dominant inheritance). In autosomal dominant disorders, affected individuals are heterozygous. The phenotype is observed in successive generations, affecting males and females equally. The offspring of an affected individual have a 50 % risk of being affected. Unaffected individuals do not transmit the disorder. In autosomal recessive disorders, affected individuals are homozygous. The phenotype tends to occur in more than one member of a sibship, affecting males and females equally. In most

233

13.2

Biochemistry and Genetics

cases, the affected individual is an offspring of two parents that both are unaffected heterozygotes. On average, one quarter of a sibship is affected. Matings between an affected individual and an unaffected unrelated partner produce only unaffected heterozygous offspring unless the partner is a carrier (unaffected heterozygote). X-linked recessive disorders are expressed mostly in males. Heterozygous females are phenotypically normal. On average, 50 % of males in a sibship are affected. They have inherited the trait from their mother. There is no father-to-son transmission. X-linked dominant disorders are expressed in successive generations. Females are affected twice as often as males. Affected males have no normal daughters and no affected sons. This pattern is rare. Y-linked inheritance concerns only males, with direct father-to-son transmission. The most important Y-linked trait is maleness. There are also infertility-causing mutations in Ylinked genes that can be transmitted to sons by intracytoplasmic sperm injection. Notice that the non-penetrant person shows no symptoms whatsoever. Mitochondrial diseases are transmitted from one affected mother to all children. All children of an affected father are normal. Sometimes, a disease-causing mitochondrial mutation is present in only part of the patient’s mitochondria. This is called heteroplasmy.

13.2. Variations of gene transmission and expression Definitions: Penetrance: The likelihood that a genotype is expressed. Many autosomal dominant diseases have incomplete penetrance, resulting in “skipped generations”. Penetrance can be quantified in percentages, or as a fraction of 1. A penetrance of 0.8 means that a person with the predisposing genotype has a likelihood of 0.8 of showing the disease phenotype Expressivity: The extent to which a genetic trait is expressed. Different patients may be either mildly or severely affected although they have the same genotype. Another phenotypic difference could be early or late age of onset. Pleiotropy: One gene results in multiple morphological, biochemical, physical, or clinical abnormalities. Locus heterogeneity: The same phenotype can be produced by mutations in different genes.

234

Functional classification of mutations

13.3.1

Allelic heterogeneity: The same phenotype can be produced by different mutations in the same gene. This is extremely common because most disease-producing mutations are loss-of-function mutations: any mutation that disrupts the function of the gene product will cause the same disease. Allelic heterogeneity usage 2: different mutations in a gene causing different phenotypes. This is seen less commonly than usage 1, but one example is the FGFR3 gene, where different mutations can cause 3 types of dwarfism, 2 types of craniosynostosis, and 1 type of skin mis-coloration. Sex-limited and sex-influenced traits: Autosomal traits that are expressed exclusively or predominantly in males or females. Example: Pattern baldness, an autosomal dominant trait expressed only in males. Anticipation: The tendency of some genetic diseases to get worse in successive generations. This is caused by an unstable “premutation” that is prone to develop into a more serious defect. The cases known so far are expansions of triplet repeats that become amplified progressively once they have reached a certain length. Imprinting: The differential expression of genetic traits depending on the maternal or paternal origin of the gene. The mechanism is incompletely understood but one of the important players is increased DNA methylation in the copy of a gene inherited from one of the parents. Contiguous gene disorders: Diseases that are caused by a large deletion (“microdeletion”, for the cytologist) that removes a group of contiguous genes. They often combine the signs of two or more single-gene disorders. Example: Wilms tumor/aniridia. Phenocopies: Conditions that resemble a genetic disease but are caused by non-genetic factors, for example deafness after intrauterine rubella infection. New mutations are common in dominant and X-linked recessive diseases, but autosomal recessive diseases are almost always inherited. The recurrence risk after the birth of a child with a new mutation is low, but above the population incidence because of possible germline mosaicism. Undisclosed nonpaternity has to be expected for about 10 % of all children.

13.3. Functional classification of mutations Medical genetics is concerned with the functional consequences of human mutations. When considering molecular pathogenesis of human mutations, different effects can be recognized.

235

13.3.2

Biochemistry and Genetics

13.3.1. Loss of function A mutation associated with a reduction or a complete loss of one or more of the normal functions of a protein is described as a loss of function mutation. Most mutations other than missense mutation result in a loss of function effect. Loss of function can be associated with either dominant or recessive inheritance, but most inborn errors of metabolism are caused by loss of function mutations which are harmless in the heterozygous state (carrier). This indicates that 50 % of normal enzyme activity is usually enough for normal function. Haploinsufficiency Loss of function mutation in the heterozygous state in which half of the normal level results in phenotypic effects are termed haploinsufficiency mutations. The phenotypic manifestations sensitive to gene dosage are a result of mutations occurring in genes that code for either receptors or more rarely enzymes, the functions of which are rate limiting. Examples of “haploinsufficiency” are familial hypercholesterolemia and acute intermittent porphyrias. There are number of autosomal dominant disorders where the mutational basis of the functional abnormality is the result of haploinsufficiency, in which homozygous mutations result in more severe phenotypic effects. This is the case for familial hypercholesterolemia.

13.3.2. Gain of function A mutation associated with an increase in one or more of the normal function of protein is described as a gain of function mutation. These are less common than loss of function mutations. For example in Huntington disease the mutant protein forms cellular aggregates which have a neurotoxic effect. In achondroplasia and thanatophoric dysplasia mutations in the FGFR3 gene result in a increased cell receptor activity leading to mild and severe suppression of bone growth. Mutations exerting gain of function usually cause conditions that show autosomal dominant inheritance. Dominant negative effect A mutation has a dominant negative effect if the product of the mutant allele interferes with the product of another allele, resulting in an adverse outcome. Dominant negative effects usually arise when the protein product is multimeric, enabling the mutant protein to disrupt the function of the final product. Structural proteins such as the collagens show

236

Linked Markers for Genotype Prediction

13.5

dominant negative effect in for example in osteogenesis imperfecta (“brittle bone disease”). The aquaporin Aqp2 is required, amongst other things, for water re-absorbtion from primary urine in the kidney. It acts as a monomer, however it is transported in a vasopressindependent manner between the plasma-membrane and intracellular stores as tetramer. Thus mutations in Aqp2 show a dominant negative effect and lead to diabetes insipidus.

13.4. Linked Markers for Genotype Prediction Allele-specific probes and PCR can be used only if the exact mutation is known. If the gene is known but the exact mutation is not (allelic heterogeneity), allele-specific probes cannot be used unless the whole gene is scanned, for example with a specially-made DNA chip. Instead, the mutation can be tracked with linked markers. A marker is a naturally occurring variation in the chromosome, normally positioned between genes or within introns. This variation can be detected using DNA methods. RFLPs can be used, but VNTRs are better because they are more variable. The human genome project has produced a map of VNTRs spanning the whole genome. Every gene in our genome has a few of them nearby. Limitations of linkage studies: 1. You have to sample DNA from several family members. 2. There is always a chance of a crossing-over, therefore you cannot make 100 % accurate predictions. The linked marker should be as close as possible to the gene or, ideally, within an intron of the gene. 3. Linkage studies may be uninformative, especially if you can get DNA of only a few family members or the marker is not sufficiently polymorphic in the family. Linkage studies can be done with Southern blotting or PCR. The length of the restriction fragment or the PCR product reflects the repeat number of the VNTR. Note that in linkage studies a VNTR allele that is linked to a disease gene in one family may be linked to the wild-type allele in another. If, in a population, a VNTR variant is more often associated with the mutant allele than expected by chance, we call this linkage disequilibrium. It suggests that the mutation occurred recently on a chromosome that happened to carry this particular variant. A constellation of two or more closely linked genetic markers is called a haplotype. In the absence of crossing-over, the gene combination of the haplotype is inherited as one unit, like a single gene.

237

occurred recently on a chromosome that happened to carry this particular Biochemistry andorGenetics variant. A constellation of two more closely linked genetic markers is called a haplotype. In the absence of crossing-over, the gene combination of the haplotype is inherited as one unit, like a single gene.

13.5

13.5. Pedigree Analysis IV.

PEDIGREE ANALYSIS

Autosomal dominant inheritance:

Autosomal dominant inheritance: - If a parent is affected, each child has a 50% risk of inheriting the disease gene.the children of unaffected individuals are not at risk. • If penetrance is complete, - If penetrance is complete, the children of unaffected individuals are not • If a patient has no affected at risk.parent, he has a new mutation (assuming complete penetrance). His own havehas a 50no % risk, although further children of his parents (assuming - children If a patient affected parent, he has a new mutation have a low risk. complete penetrance). His own children have a 50% risk, although further children of his parents have a low risk. • If a parent is affected, each child has a 50 % risk of inheriting the disease gene.

Autosomal recessive inheritance:

Autosomal recessive inheritance: • When analyzing the pedigree, start with the patient and go up and down the family When analyzing the pedigree, start with the patient and go up and tree. down the family tree.

• Parents and children- of a patient arechildren obligatory carriers. you go up carriers. and down When you Parents and of a patientWhen are obligatory the family tree, the carrier riskand is cut in half eachtree, generation. go up down thewith family the carrier risk is cut in half with

each generation.

• Most patients are produced by matings two by carriers: 1/4 between affected, 1/4 - Most patients arebetween produced matings two hocarriers: 1/4 mozygous normal, 1/2 carriers. sibling of an affected, is 2/3 risk For of being affected,For 1/4a homozygous normal,there 1/2 carriers. a sibling of an a carrier if an unaffected status isthere known. affected, is 2/3 risk of being a carrier if an unaffected status is

known. in patients. • New mutations are uncommon

- New mutations are uncommon in patients.

• If the disease is rare,- individuals outside are most likely homozygous nor-most likely If the disease is the rare,family individuals outside the family are mal. For a more accurate prediction ofnormal. the disease you accurate have to know the carrier homozygous Forrisk, a more prediction of the disease frequency in the population. risk, you have to know the carrier frequency in the population.

Probability of carriers, assuming that outsiders are homozygous normal:

Probability of carriers, assuming that outsiders are homozygous normal:

1/4 1/2

1/4 1/2 1/2

1/4

2/3 1/3 1/2 1/6 1/4

238

88

X-linked recessive inheritance 13.5.1 Probability of affected child (both parents unaffected): Probability (father is carrier) x Probability (mother is carrier) x (1/4) Probability of affected child (both parents unaffected): Probability (father is carrier) x Example: frequency in Example: the population is frequency 2%. Probability (mothercarrier is carrier) x (1/4) carrier in the population is 2 %.

Disease risk: 2/3 x 1/50 x 1/4 = 1/300 1/50

2/3

Probability of affected child if one parent is affected: (1/2) x Probability (unaffected parent is a carrier) Probability of affected child if one parent is affected: (1/2) x Probability (unaffected parent is a carrier) Probability of an affected child if both parents are affected: 100%, except in cases of locus heterogeneity where the children are unaffected double heterozygotes. Probability of an affected child if both parents are affected: 100 %, except in cases of locus heterogeneity where the children are unaffected double heterozygotes. X-linked recessive inheritance: The disease gene is passed on with the X-chromosome: From the mother to sons and daughters, from the father to daughters but not sons. - Unaffected males don’t carry the gene. 13.5.1. X-linked recessive inheritance - The patient’s mother is an obligatory carrier, unless the patient has a new mutation. - Going down the family tree in the female line, the carrier risk is cut in The disease gene ishalf passed with the X-chromosome: From the mother to sons and daughwith on each generation. ters, from the father to daughters but not sons. - All daughters of an affected father and half of the daughters of a carrier mother are carriers. - Riskmales of an don’t affected son: • Unaffected carry theOne-half gene. of the probability that the mother is a carrier. - For carrier mother with offspring of unknown sex, risk is 1/2 for • The patient’smother motherpassing is an obligatory carrier, a new is mutation. the bad allele on,unless times the 1/2 patient that thehas offspring a son (daughter does not become affected, only carriers). New mutations for many recessive diseases. • Going down the family are treecommon in the female line,X-linked the carrier risk is cut in half with each generation. Probability of carriers: • All daughters of an affected father and half of the daughters of a carrier mother are carriers.

• Risk of an affected son: One-half of the probability that the mother is a carrier. 1/2 • For carrier 1/4 mother with offspring of unknown sex, risk is 1/2 for mother passing the bad allele on, times 1/2 that the offspring is0 a son (daughter does not become affected, only1/8 carriers).

• New mutations are common for many X-linked recessive diseases. 89 239

a son (daughter does not become affected, only carriers). - New mutations are common for many X-linked recessive disea 13.6

Biochemistry and Genetics

Probability carriers: Probability ofofcarriers:

1/2 1/4 0

V. MODIFIED RISK: BAYES THEOREM. 1/8

The carrier probabilities, as determined by pedigree analysis, may have to modified. Procedure: Risk: BayestoTheorem 1. Determine13.6. the Modified original probability be or not 89 to be a carrier. This is e prior probability. The carrier probabilities, as determined by pedigree analysis, may have to be modified. 2. DetermineProcedure: the probability of the observed situation if the proband is a rrier or not a carrier.• Determine This is the theoriginal conditional probability. write probability to be or not to be aAnother carrier. Thisway is theto prior probability. s is probability(observation ⏐ genotype) 3. Multiply the •prior probability with theobserved conditional Determine the probability of the situation ifprobability, the proband is aseparately carrier or not a carrier. This is the conditional probability. Another way to write this joint is each assumption (to be or not to be a carrier). This yields the probability(observation k genotype) obability. • Multiply the prior probability with the conditional probability, separately for each 4. Compare the joint for the assumption that the proband is a carrier with assumption (to be or not to be a carrier). This yields the joint probability. e joint probability for the assumption that he/she is not a carrier. • Compare the joint for the assumption that the proband is a carrier with the joint probability for the assumption that he/she is not a carrier.

ese calculations are best done in the form of a table. These calculations are best done in the form of a table. I II 1

2

3

ample: A woman has a brother with hemophilia. She has 4 healthy sons and affected children.240How likely is it that II-2 carries the hemophilia gene? II-2 is a carrier II-2 is not a carrier Sum 1/2 1/2 ob. 1/2 x 1/2 x 1/2 x 1/2 = 1/16 1 1/2 x 1/16 = 1/32 1/2 x 1= 1/2 = 16/32 17/32

13.7

Objectives in Summary

Example: A woman has a brother with hemophilia. She has 4 healthy sons and no affected children. How likely is it that II-2 carries the hemophilia gene? prior probability conditional probability joint probability resulting probability

1/2 1/2 × 1/2 × 1/2 × 1/2 = 1/16 1/2 × 1/16 = 1/32

1/2 1 1/2 × 1 = 1/2 = 16/32 16/17

Sum

17/32

Compare the joint probabilities: 1 chance that she is a carrier : 16 chances that she is not a carrier. The carrier probability is 1 in 17 (about 6 %). [A more mathematical explanation: the two joint probabilities do not sum to one, which they really have to do (if you have only two possibilities, one or the other has to happen). Therefore we divide by the sum and get to the values in resulting probability, and now the two numbers add to one.] In this example, the prior probability is the original probability that II2 has inherited the gene. The conditional probability is the probability that all 4 sons are healthy, assuming that she is a carrier or is not a carrier. In other cases, the conditional probability is determined by the outcome of a laboratory test (how likely is it that the carrier test is negative even though the proband is actually a carrier?). Most laboratory tests have false negatives and false positives! Or it is determined by the patient’s health in a disorder with incomplete penetrance or age-dependent onset (how likely is it that the patient is healthy at his age even though he carries the disease gene?). Computers are better than physicians at diagnosing diseases because they master Bayes theorem!

13.7. Objectives in Summary 1. Recognize the patterns of Mendelian inheritance from pedigree charts. 2. Predict the carrier probabilities in pedigrees with autosomal recessive and X-linked recessive diseases. 3. Calculate disease risk in families with established Mendelian disorders. 4. Use Bayes’ theorem for the calculation of modified risk for Mendelian disorders. 5. Define the terms penetrance, expressivity, pleiotropy, locus heterogeneity, allelic heterogeneity, phenocopy and anticipation.

241

14. Krebs- (TCA-) cycle: Burning the carbon skeleton 14.1. The Pyruvate Dehydrogenase Reaction Pyruvate enters the mitochondrion on a specific carrier in the inner membrane.

14.1.1. The pyruvate dehydrogenase complex Pyruvate dehydrogenase is a multi-enzyme complex in the mitochondrial matrix. It contains 3 components, each with its own prosthetic group: • Pyruvate dehydrogenase (thiamine pyrophosphate). • Dihydrolipoyl transacetylase (lipoic acid). • Dihydrolipoyl dehydrogenase (FAD). NAD+ and CoA are soluble cofactors.

14.1.2. Overall reaction Pyruvate + NAD+ + CoA-SH → Acetyl-CoA + NADH + H+ + CO2 The reaction is irreversible. There is no way to convert acetyl-CoA back to pyruvate.

14.1.3. Functional impairment • Vitamin deficiencies, especially thiamine. • Arsenic poisoning: Arsenite binds tightly to lipoic acid. • Partial genetic deficiencies, with lactic acidosis, encephalopathy, optical atrophy, spinocerebellar ataxia. The nervous system is very sensitive to impairments of pyruvate dehydrogenase because it depends on glucose oxidation for its energy.

243

14.2.3

Biochemistry and Genetics

14.2. The TCA Cycle 14.2.1. Reactions The reaction sequence is summarized on page 308 of the Meisenberg & Simmons book. The enzymes are soluble in the mitochondrial matrix except succinate dehydrogenase (SDH) which is in the inner mitochondrial membrane. Generally, all cells that have mitochondria also have a TCA cycle.

14.2.2. Products of the TCA cycle The TCA cycle produces 2 CO2 , 3 NADH + H+ , 1 GTP and 1 FADH2 . Effectively, the 2 carbons of the acetyl residue in acetyl-CoA are oxidized to CO2 . The GTP, formed by substrate-level phosphorylation, is energetically equivalent to ATP. The most important products are NADH + H+ and FADH2 which feed into the respiratory chain.

14.2.3. Regulation of pyruvate dehydrogenase and TCA cycle • Many enzymes in the oxidative mitochondrial pathways are subject to feedback inhibition by NADH + H+ and ATP. • Irreversible reactions are catalyzed by – Pyruvate dehydrogenase – Citrate synthase – Isocitrate dehydrogenase – α-ketoglutarate dehydrogenase • Pyruvate dehydrogenase is – inhibited by its products acetyl-CoA and NADH + H+ . – inhibited by high and activated by low energy charge – inactivated by phosphorylation. – Phosphorylation is stimulated by high [ATP] / [ADP], [Acetyl-CoA] / [CoA], and [NADH + H+ ] / [NAD+ ] ratios. • Citrate synthase is inhibited by ATP and citrate.

244

Other reactions of TCA cycle intermediates

14.5

• Isocitrate dehydrogenase is stimulated by ADP and inhibited by a high [NADH + H+ ] / [NAD+ ] ratio. • α-Ketoglutarate dehydrogenase is inhibited by its products NADH + H+ and succinylCoA, and by high energy charge (note similarities to pyruvate dehydrogenase!)

14.3. Inhibition of the TCA cycle Fluoroacetate is converted enzymatically first to fluoroacetyl-CoA and then to fluorocitrate. Fluorocitrate inhibits aconitase.

14.3.1. Other reactions of TCA cycle intermediates Biosynthetic reactions α-Ketoglutarate and oxaloacetate are both precursors and degradation products of glutamate and aspartate, respectively. Succinyl-CoA is a precursor in the biosynthesis of heme. Oxaloacetate can be converted to glucose in the liver (gluconeogenesis).

Anaplerotic reactions Anaplerotic reactions produce TCA cycle intermediates. They are required because oxaloacetate has to be present to ensure continued functioning of the cycle, even when TCA cycle intermediates are drained off for biosynthesis. Most amino acids are degraded to TCA cycle intermediates. Pyruvate carboxylase produces oxaloacetate from pyruvate: pyruvate Pyruvate + CO2 + ATP + H2 O GGGGGGGGGGGGGA Oxaloacetate + ADP + Pi carboxylase The enzyme contains biotin. All ATP-dependent carboxylations require biotin. Pyruvate carboxylase is also important in gluconeogenesis. It is activated by acetyl-CoA.

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Biochemistry and Genetics

14.4. Shuttles Across the Inner Mitochondrial Membrane

14.5. Shuttles for electrons from cytoplasmic NADH + H+

Most of the NADH + H+ and FADH2 that fuel the respiratory chain are made in the mitochondrion itself: pyruvate dehydrogenase, TCA cycle, β-oxidation of fatty acids. NADH + H+ is not transported across the inner mitochondrial membrane. Electrons from cytoplasmic NADH + H+ can be channeled into the respiratory chain by the glycerol phosphate shuttle: Cytoplasm

Membrane

Mitochondrial Matirix

Pyruvate NADH, H+

FADH2

Q

H2C OH O O C O P O H2 O Dihydroxyacetone-phosphate

Glycolysis

FAD NAD+

QH2

H2C OH HO

O

C O P O H2 O Glycerol-phosphate

Glucose Glycerol phosphate is reoxidized to dihydroxyacetone phosphate by a flavoprotein in the inner mitochondrial membrane, without ever entering the Glycerol phosphate ismatrix. re-oxidized dihydroxyacetone phosphate by a flavoprotein in the mitochondrial Thistoflavoprotein transfers the hydrogen to coenzyme Q inner mitochondrial membrane, without ever entering the mitochondrial matrix. This flavo(ubiquinone), a component of the respiratory chain. The P/O ratio is 2 (2 ATP proteinformed transfers hydrogen to coenzyme Q (ubiquinone), a component of the respiratory perthe FADH 2). chain. The P/O is 2 (2 ATP formed per FADH ). In ratio the malate-aspartate shuttle, the 2hydrogen is transported into the mitochondrial matrix as malate. Because oxaloacetate is not transported across the membrane, the carbons of malate are shuttled back into the cytoplasm as aspartate: In the malate-aspartate shuttle, the hydrogen is transported into the mitochondrial matrix Mitochondrial as malate. Because oxaloacetate Cytoplasm is not transported across the membrane, the carbons of Matrix malate are shuttled back into the cytoplasm as aspartate: Pyruvate NADH, H+

Oxaloacetate

Oxaloacetate

NADH, H+

246 Glycolysis NAD+

Malate

Malate

Glucose α-Ketoglutarate

α-Ketoglutarate

NAD+

mitochondrial matrix. This flavoprotein transfers the hydrogen to coenzyme Q (ubiquinone), a component of the respiratory chain. The P/O ratio is 2 (2 ATP formed per FADH2). In the malate-aspartate shuttle, the hydrogen is transported into the mitochondrial matrix as malate. Objectives Because oxaloacetate is not transported across 14.6 the membrane, the carbons of malate are shuttled back into the cytoplasm as aspartate: Cytoplasm Mitochondrial Matrix Pyruvate NADH, H+

Oxaloacetate

Oxaloacetate

NADH, H+

Glycolysis NAD+

Malate

Malate

NAD+

Glucose α-Ketoglutarate Glutamate

Aspartate

α-Ketoglutarate Glutamate

Aspartate

14.5.1. Other substrates and products 121 Most TCA cycle intermediates are transported across the inner mitochondrial membrane by antiport carriers (‘translocases’). Also ATP, ADP, and inorganic phosphate are transported. Acetyl-CoA, oxaloacetate and NAD+ are not transported. O2 is diffusible (no carrier required).

14.6. Objectives 1. Describe the subcellular location of pyruvate dehydrogenase, its coenzyme requirements, and the balance of the reaction. 2. Predict the effects of pyruvate dehydrogenase inhibition with respect to substrate accumulation and oxygen system involvement. 3. List the sequence of intermediates in the TCA cycle and identify those reactions that produce important products like NADH + H+ , FADH2 and GTP. 4. Give reasons why anaplerotic reactions are sometimes required to maintain the TCA cycle.

247

15. Respiratory Chain, Oxidative Phosphorylation and Reactive Oxygen Species 15.1. Respiratory Chain: Burning Hydrogen 15.1.1. Overall reactions The overall reactions in the respiratory chain are: FADH2 + 1/2 O2 → FAD + H2 O ∆G00 = −200 kJ/mol NADH + H+ + 1/2 O2 → NAD+ + H2 O ∆G00 = −219 kJ/mol Actually, the electrons of the reduced coenzymes are channeled through a bucket brigade of electron carriers which form the respiratory chain. The exergonic redox reactions in the respiratory chain are used to create a proton gradient across the inner mitochondrial membrane. The dissipation of this proton gradient is coupled to the synthesis of ATP by oxidative phosphorylation.

15.1.2. The redox potential Redox reactions are 2-substrate reactions in which electrons are transferred from one substrate to the other. The tendency for a reduced substrate to donate electrons is expressed as its standard redox potential (E 0 or E 00 ), which is measured in volts. A low E 00 means high “reducing power”. Under standard conditions, electrons are transferred from the substrate with lower E 00 to the one with higher E 00 . E 00 is driving force of the reaction: 00 00 ∆E 00 = Eoxidant − Ereductant

(15.1)

∆E 00 is not a property of a redox couple but of the reaction. It is related to ∆G00 : ∆G00 = −n × F × ∆E 00 with n = number of electrons transferred and F = Faraday constant.

249

(15.2)

15.1.3

Biochemistry and Genetics

15.1.3. Components of the electron transport chain

• Flavoproteins in the respiratory chain contain FMN. • Iron-sulfur proteins contain non-heme iron bound to cysteine SH-groups, also H2 S. Fe is reversibly oxidized to Fe3+ and reduced to Fe2+ . • Cytochromes contain an iron-porphyrin as prosthetic group, either heme (Cyt. b, c, c1) or heme a (Cyt a, a3). Cyt a3 also contains (non-heme) copper. The heme iron transfers electrons by reversibly changing between the Fe2+ and Fe3+ forms. • Ubiquinone (coenzyme Q) is a hydrophobic coenzyme which is mobile in the membrane. Structure: H+ e-

O

H+ e-

O

OH

H3C O

O CH3

H3C O

O CH3

H3C O

H3C O

CH3

H3C O

CH3

H3C O

O Ubiquinon (Q)

OH Semiquinone radical (QH*)

O CH3 CH3 OH

Ubiquinol (QH2)

• Organization of the respiratory chain: Soluble components of the respiratory chain are NAD+ /NADH + H+ : The most important substrate. Ubiquinone: In the lipid phase of the membrane. Cytochrome c: A peripheral membrane protein. Molecular oxygen (O2 ): Terminal electron acceptor.

All other components are integral membrane proteins. Direction of electron flow is shown below (left to right):

250

Phosphorylation sites

15.2.2

Redox potential of some components: Redox-pair E 00 (mV) + + NAD /NADH + H -320 Ubiquinone/Ubiquinol +100 Cytochrome c Fe3+ /Fe2+ +220 1/2 O2 + 2 H+ / H2 O +820

15.2. Making ATP from electricity 15.2.1. Phosphorylation sites Oxidative phosphorylation produces 3 ATP per NADH + H+ , and 2 ATP per FADH2 : the P/O ratios are 3 and 2, respectively. The P/O ratio is the number of high-energy phosphate bonds synthesized per 1/2 O2 consumed. Electron flow through the following sites results in proton gradient formation, which can be used for phosphorylation: • NADH + H+ - Q reductase • QH2 - Cytochrome c reductase • Cytochrome oxidase Electron flow and phosphorylation are tightly coupled. Electrons will not flow through the respiratory chain unless ATP can be formed from ADP and inorganic phosphate.

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15.2.5

Biochemistry and Genetics

15.2.2. Mechanism of oxidative phosphorylation 1. The redox reactions in the respiratory chain are used to pump protons out of the mitochondrial matrix. This creates a proton gradient of 1 pH unit, and it helps to maintain a membrane potential of 200 mV, positive outside. 2. Protons move back into the mitochondrial matrix through a proton channel (Fo component) which is coupled to an ATP-synthesizing enzyme (F1 component).

15.2.3. Energy yield from glucose Anaerobically (Glucose → 2 Lactate): 2 ATP. Aerobically (Glucose + 6 O2 → 6 CO2 + 6 H2 O): Pathway Intermediate ATP Glycolysis 2 + 2 NADH + H 6 Pyruvate dehydrogenase: 2 NADH + H+ 6 TCA-cycle: 2 GTP 2 6 NADH + H+ 18 2 FADH2 4 38 The actual energy yield of oxidative metabolism is closer to 30 ATP because the substrate shuttles in the inner mitochondrial membrane require energy (they dissipate the membrane potential).

15.2.4. Regulation of electron flow and phosphorylation Required are: reduced coenzymes, oxygen, inorganic phosphate, and ADP. ADP is ratelimiting under most conditions.

15.2.5. Inhibition of oxidative phosphorylation • Inhibitors of electron transport: Amytal, rotenone: NADH - Q reductase Antimycin A: QH2 - Cytochrome c reductase. Cyanide, azide, CO, H2 S: Cytochrome oxidase.

252

Reactive Oxygen Derivatives

15.3

• Oligomycin inhibits the Fo /F1 ATPase (= mitochondrial ATP synthase). Because electron flow and ATP synthesis are coupled, electron flow ceases as well. • Most uncouplers of oxidative phosphorylation are weak hydrophobic acids which carry protons across the membrane, thus dissipating the proton gradient. ATP synthesis stops but electron flow is accelerated. Examples: 2,4-dinitrophenol, pentachlorophenol. Valinomycin uncouples by transporting potassium ions across the inner mitochondrial membrane, thus dissipating its membrane potential. • A complete lack of oxygen (anoxia) may deplete cellular ATP stores within minutes. Anoxia and hypoxia (relative lack of oxygen) are most commonly caused by ischemia (interruption of blood supply). Cell death results after a few minutes (neurons), about an hour (myocardium, liver, kidney), or several hours (fibroblasts, epidermis, skeletal muscle). Typical effects: – Decreased energy charge – Increased glycolysis, mostly from stored glycogen – Decreased pH because of lactic acid formation – Impaired ion pumping. Osmotic imbalance with cellular edema (swelling) and mitochondrial swelling. – Increased membrane permeability, both plasma membrane and organelles. – Release of lysosomal enzymes, autolysis.

15.3. Reactive Oxygen Derivatives The partial reduction of O2 , by less than 4 electrons at a time, leads to highly reactive and toxic products: Superoxide radical O2 + e− → O−· 2 + · Hydroperoxide radical O−· 2 + H → HO2

Hydrogen peroxide 2 HO·2 → H2 O2 + O2 Hydroxy radical H2 O2 * ) 2 HO· , (Fenton-reaction) Hypochlorite H2 O2 + Cl− → H2 O + OCl− Radical: Compound with an unpaired electron (indicated by · ), very reactive (“molecular terrorist”)! Superoxide radical is probably formed as a byproduct of many oxygenase reactions, but most of it is derived from the respiratory chain.

253

15.3.1

Biochemistry and Genetics

Sources of H2 O2 • Nonenzymatic formation from the superoxide radical, or directly from cytochrome oxidase. • Flavoproteins outside the mitochondria form H2 O2 . Flavoproteins in the inner mitochondrial membrane (SDH, mitochondrial glycerol phosphate dehydrogenase) transfer their electrons (hydrogen) to ubiquinone: Substrate (Reduced) Substrate (Reduced)

Product (Oxidized) Product (Oxidized)

FAD

FADH2

FAD

FADH2

QH2 QH2 1/2 O2

Q Respiratory Chain Respiratory Chain

Q H2O

H2O burst of macrophages by the 1/2 O2 H2O2 is also formed in the respiratory c) action of NADH oxidase. • H2 O2 is also formed in the respiratory burst of macrophages by the action of NADH c) + H2O2 is also formed in the respiratory burst of macrophages by the +Nonmitochondrial H oxidase. action of NADH oxidase. regenerate their prosthetic group by a flavoproteins reaction with molecular oxygen: Nonmitochondrial flavoproteins regenerate their their prosthetic group by a • Non-mitochondrial flavoproteins regenerate prosthetic group by a reaction with reaction with molecular oxygen: Substrate Product molecular oxygen: (Reduced) (Oxidized) Substrate Product (Reduced) (Oxidized) FAD

FADH2

FAD

FADH2

H2O2

O2

O2oxygen products can induce H2O2 Through free-radical chain reactions, reactive the nonenzymatic oxidation of polyunsaturated fatty acids and, probably, Through free-radical chain reactions, oxygen products can induce mutagenesis. This may be important in reactive aging, degenerative diseases, and the nonenzymatic of polyunsaturated fatty acids and, Protective probably, carcinogenesis. It oxidation is chain also responsible for acuteoxygen oxygen toxicity. Through free-radical reactions, reactive products can induce the nonenzymatic mutagenesis. This may be important in aging, degenerative diseases, and mechanisms: oxidation of polyunsaturated fatty acids and, probably, mutagenesis. This may be important carcinogenesis. It is alsodismutase responsible isfor aacute oxygen enzyme, toxicity. Protective a) Superoxide ubiquitous both in inmechanisms: aging, degenerative diseases, and carcinogenesis. It is alsoorganisms, responsible for acute oxygen mitochondria and cytoplasm. It is present in all aerobic Superoxide dismutase is aReaction: ubiquitous enzyme, both in toxicity.a) but not in obligatory anaerobes. mitochondria and cytoplasm. It is present in all aerobic organisms, but anaerobes. 2 O2not ¯ +in 2obligatory H+ O2 + Reaction: H2O2 +

2 O2¯ + 2ismechanisms H H2Otissues. 2 +most 2 Catalase found in blood O and 15.3.1.b) Protective b)

254

It is most abundant in peroxisomes. Reaction: Catalase is found in blood and most tissues. It is most abundant in peroxisomes. Reaction: 126 126

Objectives

15.4

Superoxide dismutase is a ubiquitous enzyme, both in mitochondria and cytoplasm. It is present in all aerobic organisms, but not in obligatory anaerobes. Reaction: + 2 O− 2 + 2 H → O2 + H2 O2

Catalase is found in blood and most tissues. It is most abundant in peroxisomes. Reaction: 2 H 2 O2 → 2 H 2 O + O 2 Peroxidases are enzymes that destroy hydrogen peroxide by reacting it with an organic substrate. Some peroxidases contain the rare amino acid selenocysteine in their reaction center. Many organic molecules protect against oxidative damage by reacting spontaneously with free radicals to terminate free-radical chain reactions. They include vitamin E, ascorbate, the retinoids, uric acid, and bilirubin. Proteins also protect against free radical generation by binding to metals such as iron (ferritin) and copper and zinc (metallothioneins). The free-ion form of these metals is maintained by these storage proteins at extremely low concentrations to prevent free radical damage.

15.4. Objectives 1. Define the terms “oxidation” and “reduction”. State, in qualitative terms, the relationship between the change in standard redox potential and the free NAD+ ratio for the regulation of the major energy-producing catabolic pathways. 2. State the ATP yield and energetic efficiency of glucose oxidation. 3. List examples of site- energy change of redox reaction. 4. List the major types of hydrogen and electron carriers in the respiratory chain and describe the functional organization and subcellular location of the respiratory chain. 5. Know the P/O ratios for the respiratory chain oxidation of NADH + H+ and FADH2 . 6. Describe the mechanism of oxidative phosphorylation according to the chemosmotic hypothesis. 7. Identify the limiting factors for the rate of oxidative phosphorylation in different physiological states.

255

15.4

Biochemistry and Genetics

8. State the roles of energy charge and NADH + H+ /specific inhibitors and uncouplers of oxidative phosphorylation. Predict the effects of these inhibitors and uncouplers on cellular energy charge, NADH + H+ /NAD+ ratio, thermogenesis, glycolytic activity and lactic acid formation.

256

16. Single-Gene Disorders and Traits Several thousand single-gene disorders have been described. Most are rare, with population incidences on the order of 1 in 10 000 or less. They affect at least 1 % of all newborns. Most of these diseases are autosomal dominant or autosomal recessive, but 10 % are Xlinked recessive. The molecular defect is known for most of the more important single-gene disorders. Also some harmless traits (for example eye color) are single-gene, and there are many normal polymorphisms with mendelian inheritance, for example blood groups.

16.1. Skeletal and Connective Tissue Diseases Osteogenesis Imperfecta This clinical entity is marked by brittle bones, blue sclera, and conductive hearing loss later in life. There is a wide range of severity, from perinatal lethal to mildly affected, with or without dwarfism and deformities. Inheritance: Usually autosomal dominant, rarely recessive. Molecular defect: >80 different mutations in type I collagen genes are known, most of them point mutations replacing a glycine by another amino acid. Molecular diagnosis: Linked markers can be used for prenatal and pre-symptomatic diagnosis in many families. Recurrence risk for the perinatal lethal form is 3 % (germline mosaicism!), and ultrasound monitoring of subsequent pregnancies is recommended. Ehlers-Danlos Syndrome This is a heterogeneous group of diseases with stretchy skin and loose joints. Easy bruisability and cigarette paper scars occur in most types. The three most common types are inherited as autosomal dominant traits but X-linked and autosomal recessive forms are known; overall incidence is 1 in 150 000. Marfan Syndrome There are 3 kinds of abnormality: • Long limbs, arachnodactyly. • Ocular abnormalities, with short-sightedness and/or ectopia lentis. • Aneurysm of the ascending aorta, mitral valve defects, aortic dissection.

257

16.2

Biochemistry and Genetics

Expressivity is highly variable. Phenocopies are common, and the syndrome may be confused with other genetic disorders (homocysteinuria, Ehlers-Danlos syndrome). Molecular defect: Mutations in the gene for the connective tissue protein fibrillin. Molecular diagnosis: There is much allelic heterogeneity. Therefore linked markers are most commonly used. Treatment: Avoidance of vigorous exercise and aggressive treatment of hypertension.

16.2. Skeletal Dysplasias and Dysostoses Skeletal dysplasias are disorders with abnormal body proportions, frequently dwarfism. Dysostoses are abnormalities of individual bones or groups of bones. > 100 different dysplasias have been described, with different modes of inheritance. Examples: Apert syndrome is a malformation syndrome with craniosynostosis (abnormal fusion of cranial sutures), syndactyly, cardiac septation defects (atrial or ventricular), progressive hearing loss, delayed development, mental deficiency, and increased sweat production. Caused by new mutations in the gene for a fibroblast growth factor receptor (FGFR2). Incidence 1 in 10 000. Achondroplasia is the most common form of short-limbed dwarfism. Incidence is 1 in 10 000 even though 7/8 are caused by a new mutation. Height 120–135 cm with marked shortening of the proximal parts of the extremities (rhizomelia), depressed nasal bridge, bulging forehead, and lumbar lordosis. Complete penetrance. Patients are fertile and have a normal life span. Homozygotes (some of the children of two achondroplastics) are seriously malformed and die shortly after birth. Most cases are caused by a Gly → Arg substitution in the transmembrane domain of a fibroblast growth factor receptor (FGFR3) that is expressed in chondrocytes. This abnormal receptor is constitutively active. Other FGFR3 mutations cause two other forms of dwarfism (hypochondroplasia and thanatophoric dysplasia ), two forms of craniosynostosis (Muenke and Cruzons ), and a skin coloration disorder (acanthosis nigricans). Prenatal diagnosis of achondroplasia is possible with ultrasound or allele-specific probes - but what do you do when achondroplastic parents want to use prenatal diagnosis to ensure that their child is also a dwarf? The differential diagnosis of skeletal dysplasias and dwarfism requires X-rays and other diagnostic tests. Patients should be referred to a specialist for the diagnostic workup.

258

Other Muscular Dystrophies

16.3.1

Hypophosphatemia (Vitamin D Resistant Rickets) The patients have bone demineralization and deformities as in rickets, but do not respond to normal doses of Vitamin D. This condition is caused by a renal transport defect for phosphate. It is X-linked, with incomplete penetrance in heterozygous females.

16.3. Diseases Of Muscles and Peripheral Nerves Muscular dystrophies are inherited muscle diseases with progressive muscle wasting and weakness. Differential diagnosis includes peripheral neuropathies, spinal muscular atrophies, inflammatory diseases (myositis, dermatomyositis), inborn errors of muscle metabolism, and myasthenia gravis. The muscle diseases can be distinguished from the neuropathies by elevated blood levels of creatine kinase. Other diagnostic tests are muscle biopsy, electromyography, and measurements of peripheral nerve conduction velocity. Duchenne Muscular Dystrophy (DMD) This X-linked recessive disease is the most common (1 in 4000 males) and most deadly muscular dystrophy. Patients are normal for 1 or 2 years after birth, before developing progressive muscle weakness and wasting. There is pseudohypertrophy of the calf muscles, inability to walk by age 10–12, death at 20 a (respiratory or cardiac failure), mild mental impairment, and massive creatine kinase elevation even in the pre-symptomatic stage. Molecular defect: The gene is huge, with 79 exons spread over 2.2 × 106 bp. This is the largest gene in the genome! It codes for dystrophin, a structural protein that connects the sarcolemma with the cytoskeleton. It is also present in the brain. Most mutations are large deletions. Molecular diagnosis: Deletions in patients can be detected by Southern blotting or PCR. If the nature of the mutation is unknown, linked markers can be used for carrier detection. Many carrier females have mildly elevated creatine kinase, and 8–10 % show symptoms. This is an unusually large proportion for an X-linked disease (about 5 % is a more common number). Gene therapy: Somatic gene therapy aimed at myoblasts is in the experimental stage. Technical problem: The dystrophin gene is so large (13 000 bp coding sequence) that it is hard to fit into retroviral vectors.

16.3.1. Other Muscular Dystrophies Becker muscular dystrophy: Mutations in the dystrophin gene (frameshift), but milder than Duchenne.

259

16.4.1

Biochemistry and Genetics

Limb-girdle muscular dystrophy: Autosomal recessive, juvenile or adult onset and slowly progressive, and no mental impairment. Severity varies, slower progression cf. DMD. Defect in one of the four sarcoglycan genes.

Facio-scapulo-humeral muscular dystrophy:!fascio-scapulo-humeral Autosomal dominant with variable age of onset and variable severity.

16.3.2. Myotonic Dystrophy Autosomal dominant, incidence 1 in 10 000. Weakness and wasting of facial muscles, sternomastoid and distal limb muscles, and myotonia. Also cataracts and cardiac conduction defects and endocrine changes. Severe cases with hypotonia from birth, pouting expression, and mild mental deficiency. Two forms: DM1 is caused by the amplification of a trinucleotide repeat in the 3’-untranslated region of a gene for a protein kinase (myotonin kinase). DM2 is caused by a CCTG repeat in an intron of ZNF9 (an RNA binding protein) The severe cases have only been described for DM1. Age of onset and severity depend on the repeat number. The repeat, once amplified, is unstable in female meiosis, with pronounced anticipation when inherited from the mother. PROMM is a milder phenotype (proximal myotonic myopathy) also caused by these mutations.

16.3.3. Peripheral Neuropathies Inherited motor + sensory peripheral neuropathies (HMSNs) are known as CharcotMarie-Tooth (CMT) syndrome. Inheritance is autosomal dominant, rarely autosomal recessive or X-linked recessive. Overall incidence 1 in 2500. Most common: CMT type IA. Autosomal dominant, caused by duplication of a 1.5 Mb portion of chromosome 17p. The duplicated part contains a gene for a myelin membrane protein. Peripheral nerve conduction velocity is reduced in this and some other types of CMT disease. Mild to moderate handicap with weakness of peroneal muscles and foot drop, some sensory loss, normal life span.

260

Mental Retardation

16.4.2

16.4. CNS Disorders 16.4.1. Hereditary ataxias This is a heterogeneous group, with intermittent or progressive ataxia and usually associated with other neurological problems. Friedreich’s ataxia (incidence 1 in 40 000) is an autosomal recessive disease, chronic progressive with onset usually between 4 and 20 years. There is degeneration of dorsal root ganglion cells and spinocerebellar tract, with progressive ataxia, sensory neuropathy, and areflexia. Most patients die in their 30s–50s. The disease is caused by the expansion of a GAA trinucleotide repeat in the first intron of a gene. The trinucleotide is amplified from a normal length of < 22 to > 200. Some patients have no trinucleotide expansion but an inactivating point mutation in one copy of the gene. Other ataxias: • Deficiency of pyruvate dehydrogenase or pyruvate carboxylase • Deficiencies of urea cycle enzymes. - Hartnup’s disease • Abetalipoproteinemia • Ataxia-telangiectasia, a chromosome breakage syndrome with rash immunodeficiency, and malignancies.

16.4.2. Mental Retardation Some mentally retarded patients have an identifiable single-gene disorder. Most common (1 in 2000 males): Fragile X Mental Retardation Affected males have mental retardation of variable severity. Mild dysmorphic features: long face, large ears and jaw, and large testes. Some carrier females are also mentally deficient, and some males with the mutation are not retarded (“normal transmitting males”). This disease have alternatively been described as an X-linked recessive or as a dominant disease with reduced penetrance; the proportion of manifesting carrier females have been reported at anywhere between 20 % and 50 %; manifesting carrier females generally have very long repeats, longer than the average expressing male. Molecular defect: A triplet repeat CGG in the 5’-untranslated region of a gene is amplified. Normal people have 6–54 copies of the repeat (average 29). It is amplified to 60–200 repeats in normal transmitting males and > 200 in retarded males. The large amplifications

261

16.5.1

Biochemistry and Genetics

become heavily methylated. This silences the gene. Once the sequence is amplified to > 60 copies, it becomes unstable in female meiosis, and further amplifications occur in successive generations. Very large repeats are also unstable in mitosis. Therefore, there may be extensive mosaicism (different repeat numbers in different somatic cells). Also patients with loss-of-function mutations (deletions, frameshifts) in this gene are retarded. The affected gene (FMR1) is expressed in the brain, but otherwise its function is unknown. Diagnosis: Affected males and carrier females have a fragile site in Xq27. The fragile site is seen only when cells are cultured in folate-deficient media. Southern blotting and PCR are now the preferred procedures in affected families. These methods provide a good estimate of the repeat number, but PCR generally cannot produce a product from the longer repeats. Screening by dot-blotting or microarray is technically difficult.

Huntington’s Disease This is a neurodegenerative disease with cell death in the corpus striatum and (less severe) the cerebral cortex. The first signs are vague personality changes, often with poor judgment and/or irresponsible behavior. This is followed by a motor disorder with chorea and athetosis, accompanied by progressive dementia. Most patients die 15–20 years after onset of the motor disorder. 20 % of cases are diagnosed before 26 years of age, 50 % before 35, and 80 % before 43. Penetrance is 100 % in patients living long enough. Incidence: Variable, about 1 in 20 000 in the US, 1 in 333 000 in Japan. New mutations are rare. Molecular defect: Amplification of a CAG trinucleotide repeat in the coding region of a gene (coding for glutamine). Normal people have 10–29 copies of the repeat, patients 36–120. Expansion of the repeat can occur in male meiosis. Repeat number correlates with severity and age of onset. The normal function of the gene is unknown. Diagnosis: PCR or (rarely) Southern blotting.

16.5. Blood Diseases 16.5.1. Clotting Disorders There are several inherited deficiencies of clotting factors. Most important:

262

Occulocutaneous albinism

16.6.2

Hemophilia A (“classical” hemophilia). This condition is X-linked recessive. There is not much spontaneous bleeding, but prolonged bleeding occurs after minor injuries. Repeated bleeding into joints can lead to arthritis. With modern treatment (transfusion of factor VIII during bleeding episodes), the life expectancy is only mildly reduced. Molecular defect: Many different mutations in the factor VIII gene have been identified. Diagnosis: Carrier detection by RIA of factor VIII is unreliable. Allelic heterogeneity makes the use of allele-specific methods difficult, therefore gene tracking with linked markers is used for carrier testing and prenatal diagnosis. Complementation tests can be used to differentiate hemophilia A from other bleeding disorders.

16.5.2. Structural defects of RBCs In hereditary spherocytosis and elliptocytosis, RBCs are spherical or elliptical. These RBCs have a reduced life span in vivo because they are removed by splenic macrophages, and they have increased fragility in vitro. Patients are either asymptomatic or have mild anemia. These conditions are caused by inherited abnormalities of spectrin, band 3 protein, or other components of the membrane skeleton. The spectrin content of RBCs is always reduced because any spectrin that is not tied into the membrane skeleton is degraded. Inheritance: Autosomal dominant in most patients. Diagnosis: Hematology (RBC shape). DNA probes or linkage studies are useless because of extensive heterogeneity (both locus and allelic heterogeneity). Treatment: No treatment is required in mild cases. Splenectomy is indicated in anemic patients.

16.6. Skin Diseases 16.6.1. Occulocutaneous albinism Albinos have white skin, white or yellow hair, and pink iris in typical cases. They get sunburn, are at risk for skin cancer, and may have mild visual disturbances. Inheritance: All forms are autosomal recessive. Molecular defect: Nearly half of all albinos cannot make tyrosinase (“tyrosinase-negative” albinism). Most tyrosinase-positive albinos have a mutation in a gene that codes for a membrane protein, probably a transporter for tyrosine in the melanosome membrane. Treatment: Straw hat and sunglasses.

263

16.8

Biochemistry and Genetics

16.6.2. Epidermolysis bullosa (EB) This is a group of skin blistering diseases with damage to the dermal-epidermal junction in response to mild trauma. Most forms are autosomal dominant. Most common: EB simplex, caused by defects in the genes for keratin 5 or keratin 14 in the basal layer of the epidermis.

16.7. Polycystic Kidney Disease Adult polycystic kidney disease is a relatively common (1 in 1000) autosomal dominant trait, with cyst development in kidneys, liver, pancreas and spleen. 50 % of patients develop kidney failure by age 70. 10 % of all patients with end-stage renal disease have polycystic kidney disease. Prenatal diagnosis with allele-specific probes or linked markers is possible. Renal cysts can be detected with ultrasound in pre-symptomatic patients. This disease can be caused by mutations in 2 different genes (locus heterogeneity). Infantile polycystic kidney disease is a rare autosomal recessive disease, with death in early childhood. Prenatal diagnosis is possible by ultrasound.

16.8. Cystic Fibrosis Cystic fibrosis (mucoviscidosis) is the most common lethal single-gene disorder in the white population (1 in 2000). Exocrine glands of skin, GI-tract, respiratory tract and male reproductive tract are affected, with hyperviscosity of secretions. Specific problems: • Neonatal bowel obstruction (meconium ileus) in 5–10 % of cases. • Pancreatic insufficiency (cyst formation and fibrosis), with malabsorption and steatorrhea even in babies. • Bronchial obstruction and recurrent lung infections ( Ps. aeruginosa and Staph. aureus). • Increased CI− , Na+ and K+ in sweat. Babies taste salty. • Male infertility, atresia of the vas deferens. This is a severe disease, but with good medical care many patients survive into their 40s. Pancreatic enzyme replacement, dietary management, bronchodilators, and prophylactic antibiotics are the mainstay of treatment.

264

“Harmless” Mendelian Traits

16.10

Molecular defect: The gene product is a cAMP-regulated chloride channel in exocrine glands (CFTR = cystic fibrosis transmembrane regulator). A 3-base-pair deletion is most common (∆F508 , 70 % in Northern Europe), but > 100 other mutations have been identified. Some of these mutations lead to a milder form of the disease; the mildest symptoms described include only the male infertility. Carrier detection: Molecular probes are available for the more common mutations, but not the exotic ones. Prenatal diagnosis: By PCR and/or allele-specific probes. Gene therapy: Somatic gene therapy aimed at the respiratory epithelium (using adenoviral vectors) has been attempted.

16.9. Blindness and Deafness Severe congenital deafness (deaf-mutism) occurs with a frequency of 1 in 2000. 60 % are autosomal recessive, 10 % autosomal dominant or X-linked, 30 % phenocopies. There is extensive locus heterogeneity. Recurrence risk after an affected child is born to hearing parents: 1 in 7. After two affected children: 1 in 4. Congenital blindness is rare, but several retinal diseases that lead to blindness later in life are single-gene. Most common phenotype: Retinitis pigmentosa, with night blindness, tunnel vision, blindness with variable age of onset. Overall incidence is 1 in 5000. Inheritance is autosomal dominant (15 %), autosomal recessive (70 %) or X-linked recessive (15 %). Some patients with autosomal dominant retinitis pigmentosa have mutations in the gene for rhodopsin. The most common forms of acquired blindness (macular degeneration, glaucoma, cataract, diabetic retinopathy) are not single-gene but multifactorial. Genetic counseling is difficult both for congenital deafness and for retinitis pigmentosa because of the extensive locus heterogeneity and allelic heterogeneity.

16.10. “Harmless” Mendelian Traits Some normal variations and minor “abnormal” conditions are inherited as simple Mendelian traits. Red-green color blindness This is a common X-linked recessive condition affecting 5–10 % of all males. There are two forms: Protanopia (red blindness, incidence 1 %), and deuteranopia (green blindness, incidence 5 %). Partial defects are called protanomaly (1 %) and deuteranomaly (1.5 %). The affected genes code for the red and green pigments in cones. They are close together on the X chromosome. Color blindness

265

16.12

Biochemistry and Genetics

is diagnosed with color charts. Defects of the blue pigment (coded by an autosomal gene) are rare, as is total color blindness (failure of cone development). Male pattern baldness This trait is autosomal dominant with male-limited expression in most families, with hair loss in young men, usually at 20–35 years of age. Expression is male limited because testosterone is necessary for the phenotype; a castrate will not get bald. More limited hair loss in older men is usually polygenic. Eye color There are two or three major interacting genes. In most (but not all) families, brown is dominant over gray/green. Green/blue is thought to depend on a second gene, with green dominant over blue. Cerumen (ear wax) Most Europeans and Africans have wet ear wax, but 80 % of Japanese and variable proportions of other orientals have dry ear wax. Wet ear wax is dominant over the dry type.

16.11. Tuberous Sclerosis Tuberous sclerosis is an inherited neuro-cutaneous disorder with incidence about 1/5000 in some populations that involves many organ systems. Clinical picture includes epilepsy, learning difficulties, behavioral problems, and skin lesions. One source of pathology is benign tumors of especially skin, kidney, and brain. Penetrance is high, but some of the symptoms are so subtle that only a dedicated search will detect the disease in a person; there is widely variable expressivity. Genetic background shows locus heterogeneity, at least 2 and probably 4 loci involved; the two well-characterized loci are classified as tumor suppressor genes and the gene products are involved in intracellular regulation of e.g., insulin signaling. Symptoms as stated are variable, but lowered IQ, epilepsy and seizures, skin patches with angiofibromas, hypopigmented maculaes, Shagreen patches, or a distinctive brown fibrous patch on the forehead are all commonly occurring in this disease. Renal angiomyolipoma or cysts are common. Cardiac rhabdomyoma is a cause for prenatal death (sometimes seen as more than one spontaneous abortion when one parent is affected), but if the child survives, rhabdomyomas will often spontaneously regress. A benign tumor is a problem especially if it occurs inside the skull, but sometimes the characteristic benign tumors actually develop into malignant and metastatic states.

16.12. Phenylketonuria (PKU) PKU is caused by increased levels of phenylalanine in the blood and the typical cases involve mutations in the phenylalanine hydroxylase gene (PAH) inherited in an autosomal

266

Hemochromatosis

16.13

recessive fashion. The homozygous child is normal at birth, but fail to achieve developmental milestones and develop microcephaly and progressive impairment of cerebral function. Hyperactivity, seizures and severe mental retardation are major symptoms later in life. A “mousy” odor, hypopigmentation and eczema are other findings. To completely avoid the development of symptoms, treatment must be initiated by the third week after birth; treatment consists of a special diet low in phenylalanine and high in tyrosine, and monitoring the plasma concentrations of these two amino acids. Ideally, treatment should be continued throughout life, but at least until development is complete. If a woman has been developing normally and then stopped the diet in adulthood, she needs to restart the diet well before getting pregnant; otherwise the child will be born with microcephaly and many other malformations, and after birth show severe neurodevelopmental delay and growth retardation. The incidence of PKU has traditionally been listed as 1/10 000 in medical genetics textbooks, which still might be a valid number for European Caucasians. In the USA, recent estimates for classical PKU range from 1/13 500 to 1/19 000. The disease is more common in Caucasians and Native Americans, while the incidence is lower in African Americans, Hispanics and Asian Americans (a different source mentions “Orientals” as a high risk group). Non-PKU hyperphenylalaninemia (caused by mutations in PAH with some residual enzyme activity) is somewhat rarer.

16.13. Hemochromatosis The clinical features of hemochromatosis include cirrhosis of the liver, diabetes, hypermelanotic pigmentation of the skin, and heart failure. Primary hepatocellular carcinoma, complicating cirrhosis, is responsible for about one-third of deaths in affected homozygotes. Since hemochromatosis is a relatively easily treated disorder if diagnosed, this is a form of preventable cancer. At least 5 iron-overload disorders labeled hemochromatosis have been identified on the basis of clinical, biochemical, and genetic characteristics.

Classic hemochromatosis (HFE) An autosomal recessive disorder, is most often caused by mutation in the gene for ferroportin on chromosome 6p21.3. It has also been found to be caused by mutation in the gene encoding hemojuvelin, which maps to 1q21. This form is common in Caucasians of Northern European descent, incidences of 1/200–1/300 and 1/72 in NE Quebec.

267

16.14.1

Biochemistry and Genetics

Juvenile hemochromatosis , or hemochromatosis type 2 (HFE2) Also autosomal recessive. One form, designated HFE2A, is caused by mutation in the HJV gene. A second form, designated HFE2B, is caused by mutation in the gene encoding hepcidin antimicrobial peptide, which maps to 19q13. This and the other minor forms of HFE are rare. One form is autosomal dominant. Chromium, an essential trace mineral required for normal insulin function, is transported bound to transferrin and competes with iron for that binding. It has been found that less chromium is retained in patients with hemochromatosis than in controls, suggesting that the diabetes of hemochromatosis may be due in part to chromium deficiency. Treatment of the iron overload is through phlebotomy about once a week until the iron content has gone down, and less often after. Desferrioxamine can be given if anemia develops. Iron intake should be avoided. The different symptoms should be treated as per best practice.

16.14. Diseases Caused by Unusual Mutations 16.14.1. Imprinting-related Syndromes Imprinting is not completely understood, but DNA methylation at CG sequences is involved. A highly methylated region often changes chromatin structure and transcription is turned off. More than 20 loci are known to be imprinted on one allele in normal subjects. The imprinting pattern also depends on which parent the chromosome is derived from. In some cases, a locus will be imprinted if derived from the father but not the mother, and in other cases, vice-versa. Prader-Willi syndrome is characterized by hypotonia in infants, and by obesity, hypogonadism, short stature, small hands and feet and mental retardation in children and adults. It is usually caused by deletion in the long arm of chromosome 15 inherited from the father. Angelman syndrome (“happy puppet syndrome”) includes mental retardation, ataxia and hyperactivity with jerky movements, and inappropriate laughter. It is caused by the same deletion as in Prader-Willi syndrome, but inherited from the mother. Both Prader-Willi and Angelman syndromes are caused by effects on imprinting. The Prader-Willi/Angelman region is unusual in that the region contains two imprinted regions very close together but non-overlapping. The common deletion overlapping both imprinting regions, if inherited from the mother, leads to lack of an active gene for the

268

Objectives in Summary

16.15

“Angelman gene” (ie. the E6-associated protein ubiquitin-protein ligase gene [UBE3A]). A different gene is inactive if the deletion is inherited from the father and the undeleted copy from the mother. A more unusual cause of either Angelman or Prader-Willi syndrome is uniparental disomy (5–20 % of cases)! If both copies of chromosome 15 are inherited from the father, they will have Angelman syndrome, if they are inherited from the mother it leads to Prader-Willi syndrome. Angelman caused by uniparental disomy is milder than Angelman caused by deletion. Beckwith-Wiedemann syndrome (BWS) is characterized by overgrowth, resulting in exomphalos, macroglossia, and gigantism in the neonate. Hemihypertrophy, visceromegaly, adrenocortical cytomegaly, and dysplasia of the renal medulla are additional features. Adrenal carcinoma, nephroblastoma (Wilm’s tumor), hepatoblastoma, and rhabdomyosarcoma occur with increased frequency (≈ 5 %) particularly in those with hemihypertrophy. There may be linear indentations of the ear lobe and/or peculiar posterior helical ear pits. The phenotype tends to normalize with age. In normal subjects, only the paternal copy of the growth factor IGF-2 on chromosome 11 is expressed while the maternal copy is silenced through imprinting. BSW is most often caused by loss of imprinting, leading to transcription of both copies of IGF-2.

16.14.2. Lepore Hemoglobins Some patients with β-thalassaemia have a Lepore hemoglobin: They are lacking the normal genes for β and δ chains but have a new gene instead that starts as δ and ends as β. Because it has the promoter of the δ chain gene, it is expressed at a low rate, accounting for the β-thalassaemia. Some normal individuals have an anti-Lepore mutation: There is a complete β-chain gene, a complete δ-chain gene and an abnormal gene that starts as β and ends as δ. These mutations are caused by unequal crossing-over during prophase of meiosis I. This type of mutation is important during evolution. It can not only create novel fusion genes, but also gene duplications. It is responsible for the existence of gene clusters in our genome.

16.15. Objectives in Summary 1. State the approximate incidence, the phenotypic features and the molecular lesions of the most important Mendelian disorders (Osteogenesis Imperfecta, Ehlers-Danlos Syndrome, Achondroplasia, Marfan Syndrome, Duchenne Muscular Dystrophy, Myotonic Dystrophy, Charcot-Marie-Tooth Disease, Cystic Fibrosis, Polycystic Kidney Disease, Occulocutaneous Albinism, Red-green Color Blindness, Hemophilia

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Biochemistry and Genetics

A, Hereditary Spherocytosis and Elliptocytosis, Huntington’s Disease, Friedreich’s Ataxia, Fragile X Mental Retardation, Deafness and Retinitis pigmentosa, Tuberous Sclerosis, Phenylketonuria, Hemochromatosis).

270

17. Hormone Biochemistry 17.1. Types of Extracellular Messenger The transduction of a chemical signal from cell to cell may be Endocrine: The messenger is a hormone that is transported by the blood. In most but not all cases, the hormone is formed in a specialized endocrine gland. Paracrine: The messenger acts on neighboring cells in the tissue where it is formed. Example: prostaglandins. Autocrine: The messenger is secreted, but acts on the synthesizing cell. Many paracrine messengers are also autocrine. Neurotransmission is a specialized kind of paracrine signaling in which the extracellular messenger, or neurotransmitter, is secreted by a neuron at a specialized junction called a synapse.

17.2. Hormone Receptors A hormone receptor is a cellular protein which binds the hormone non-covalently and mediates its physiological effects. It may be an integral protein of the plasma membrane, or it may be intracellular. Receptor binding is always the first step in hormone action. Hormone binding induces a conformational change in the receptor protein, and this conformational change triggers the cellular response. Receptor binding is: • High-affinity • Reversible • Specific for the hormone or class of hormones • Saturable

271

17.4

Biochemistry and Genetics

The dissociation constant KD corresponds to the hormone concentration at which half of the receptor sites are occupied. It describes the affinity between receptor and hormone. The maximal binding Bmax corresponds to the number of receptors. The presence or absence of a receptor determines whether or not a cell responds to a specific hormone. The magnitude of the response depends on the number of receptor-hormone complexes. Hormone action depends on the number of receptors (Bmax ), their affinity for the hormone (KD ), and the coupling of the receptor with its downstream targets. All of these may be regulated physiologically. Also many drugs act on receptors that were originally designed for hormones or neurotransmitters.

17.3. Types of Hormone Receptor Some extracellular messengers can freely diffuse across membranes, but most cannot. Watersoluble messengers have to bind to a receptor in the plasma membrane. Types: Steroid hormone receptors are zinc-finger proteins (see fig. 4.1 on page 93). They are located either in the nucleus or the cytoplasm of unstimulated cells. Without hormones, the receptor protein does not bind DNA. With the bound hormone, however, it regulates transcription by binding to response elements on the DNA. The receptors for thyroid hormones, retinoic acid, and vitamin D work by the same mechanism. Since effects are mediated through protein synthesis and the proteins have fairly long life spans, this type of action cannot work on a minute-by-minute basis. Neurotransmitter receptors mediating the fast actions of neurotransmitters on the membrane potential are ligand-gated ion channels. These channels are closed in the absence of the neurotransmitter, but open within a millisecond or so when the transmitter binds. The opening of sodium or calcium channels causes depolarization (excitation). Example: nicotinic acetylcholine receptor in the neuromuscular junction. The opening of chloride or potassium channels causes hyperpolarization (inhibition). Example: GABA-A receptor in the brain. Receptors with enzymatic activity are inactive in the unstimulated state, but catalyze a reaction when the extracellular messenger is bound. Example: Receptors for growth factors and insulin phosphorylate tyrosine side chains in proteins. G-protein linked receptors mediate most of the classical hormone effects. They usually trigger the formation of a second messenger.

272

Receptors Coupled to G-Protein

17.5

17.4. Receptors Coupled to G-Protein These cell surface receptors belong to the family of the 7-transmembrane receptors: they criss-cross the membrane seven times. G-proteins are attached to the hormone receptor on the cytoplasmic surface of the plasma membrane. There are several families of G-proteins which are associated with different receptors and act on different effector systems. These G-proteins have 3 subunits: α, β and γ. The β and γ subunits are closely bound to each other, but the α-subunit can easily dissociate from βγ. The α-subunit has a binding site for GTP and GDP. In the resting state, the complete heterotrimeric G-protein, with GDP bound to the α-subunit, is bound to the intracellular domain of the hormone receptor. After hormone binding: 1. The conformation of both the receptor and the attached G-protein changes. 2. This results in the dissociation of GDP from the α-subunit and its replacement by GTP. 3. The α-subunit -GTP complex dissociates from βγ. 4. Both the α-GTP complex and the βγ leave the hormone receptor and move along the inner surface of the plasma membrane. 5. α-GTP and βγ bind to effectors on the inner surface of the plasma membrane. Most effectors are enzymes for the synthesis of a second messenger, but some G-proteins act on ion channels in the plasma membrane. 6. The α-subunit has an intrinsic GTPase activity which hydrolyzes the bound GTP. GDP remains bound to the α-subunit, but the α-GDP complex no longer binds the effector. It binds to βγ and the receptor instead. 7. Upon re-stimulation of the receptor, GTP binds in exchange for GDP, and the cycle is repeated. The GTPase activity of the α-subunit is necessary to terminate hormone action. Types of G-protein: The G-proteins are classified according to the type of α-subunit they contain: Gs -protein (s = stimulatory) stimulate adenylate cyclase. Gi -proteins (i = inhibitory) inhibit adenylate cyclase. Gq -proteins stimulate a phosphatidylinositol-specific phospholipase C. G12 -proteins regulate ion channels.

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17.5

Biochemistry and Genetics

17.5. Cyclic AMP (cAMP) cAMP is formed by adenylate cyclase: O O O H2 O P O P O P O C

-

O

O

O

H2 O C

Adenine

O

Adenine

adenylate cyclase

O

(on plasma membrane)

OH

O

OH

+

O P O

PPi

OH

cAMP

ATP

The active site of adenylate cyclase is on the inner (cytoplasmic) surface of the plasma membrane and its activity is always under hormonal control. cAMP is inactivated by phosphodiesterase: O

H2 O C

O

+

O P O

O

H2 O P O C

Adenine phosphodiesterase

O

Adenine

O

H2O

+

OH

OH

H

+

OH

Several phosphodiesterases exist. Some of them are also controlled by hormones. Phosphodiesterases are inhibited by methylxanthines (caffeine, theophylline, aminophylline). The effects of cAMP are mediated by protein kinase A (PKA): Cat

cAMP Reg

Reg

Cat

Cat

+ 4 cAMP cAMP

inactive Protein kinase A

Reg

cAMP

Reg

cAMP Cat

catalytically active subunits

This enzyme phosphorylates specific proteins on serine or threonine residues, including some enzymes (glycogen synthase, phosphorylase kinase etc) and nuclear proteins. The most important nuclear targets are transcription factors of the CREB family (CREB = cAMP response element binding). CREB proteins bind to the cAMP response element. The phosphorylation of DNA-bound CREB proteins by protein kinase A stimulates transcription.

274

Calcium and Phosphatidylinositol

17.6

cAMP mediates the effects of calcitonin, PTH, TSH, epinephrine (β-receptors only), vasopressin (on kidney), glucagon, ACTH, and many other hormones. Clinical problems: • Patients with pseudohypoparathyroidism show signs of PTH deficiency (hypocalcemia, tetany,) but unlike true hypoparathyroidism, other abnormal signs (short stature, mental and neurological problems) are also present, and PTH levels are normal or elevated. Many of these patients have a defective Gs protein, with inefficient coupling of hormone receptor to adenylate cyclase. • The symptoms of cholera are induced by the exotoxin of the bacterium Vibrio cholerae. Cholera toxin binds to the surface of intestinal mucosal cells, and one of its subunits enters the cell. This subunit is an enzyme which ADP-ribosylates a specific arginine side chain in the αs -subunit of Gs : αs -subunit + NAD+ → αs -subunit-ribose-P-P-ribose-adenine + Nicotinamide This covalently modified αs -subunit can still bind GTP and activate adenylate cyclase, but it has lost its GTPase activity. This results in prolonged stimulation of adenylate cyclase. The typical diarrhea of cholera is mediated by excessive accumulation of cAMP in the intestinal mucosal cells. Also one of the toxins of enterotoxic E. coli, the cause of traveler’s diarrhea (“Montezuma’s revenge”), acts by a similar mechanism. Some hormones (endorphins, α2 -adrenergic agonists, D2 -dopamine etc.) decrease cellular cAMP levels. Their effect is mediated by an inhibitory G-protein (G1 ). Pertussis toxin (Bordetella pertussis causes whooping cough) modifies G1 covalently, thereby locking it in the inactive GDP-form.

17.6. Calcium and Phosphatidylinositol Calcium concentrations in the cytoplasm are normally very low (< 1 × 10−6 M). Some hormones and neurotransmitters, such as epinephrine (α1 -receptors), acetylcholine (muscarinic receptors), histamine (H1 -receptors), angiotensin II, serotonin, and vasopressin in blood vessels, cause the release of calcium from the ER, thereby increasing its cytoplasmic concentration. Through a G-protein, the activated hormone receptor stimulates a phosphoinositide-specific phospholipase C, which cleaves the membrane lipid phosphatidylinositol-4,5-bisphosphate into inositol -1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. IP3 diffuses to the ER membrane where it opens a calcium channel. Calcium affects cellular processes by binding to various regulatory proteins: Troponin C mediates contraction of striated muscle.

275

17.7

Biochemistry and Genetics

Calmodulin is structurally related to troponin C. Present in all nucleated cells. The calciumcalmodulin complex activates many enzymes, including phosphorylase kinase, the calcium pump in the sarcoplasmic reticulum, brain phosphodiesterase, and myosinlight-chain kinase in smooth muscle. Protein kinase C (PKC) is attached to the inner surface of the plasma membrane, with substrate specificity different from protein kinase A. Activated by calcium, diacylglycerol (a product of phospholipase C!) and phospholipids. There are many isoenzymes of protein kinase C with different regulatory properties. Pharmacologically, protein kinase C is activated by phorbol esters (from croton oil), which act as tumor promoters. Muscle contraction is always triggered by calcium. In striated muscle, it is mediated by troponin C. Smooth muscle has no troponin, but calcium binds to calmodulin and this complex stimulates myosin light chain kinase. The phosphorylation of the myosin light chains by this enzyme causes contraction. cAMP opposes the calcium effect in smooth muscle. In vascular smooth muscle, α1 -adrenergic agonists, vasopressin and angiotensin contract by increasing calcium, β-adrenergic agonists relax by increasing cAMP. In bronchial smooth muscle, histamine (H1 receptor) and acetylcholine (muscarinic receptor) contract by increasing Ca2+ , β-adrenergic agonists relax by increasing cAMP. Phosphodiesterase inhibitors (theophylline, aminophylline) are good for asthma because they increase cAMP. Exocytosis of water soluble products from storage vesicles in secretory cells is triggered by calcium: transmitters from nerve endings, histamine from mast cells, insulin from β-cells, zymogens from the exocrine pancreas. Cell growth and/or mitosis is often stimulated by the phospholipase (C)-(IP3)-calcium system. Besides the phospholipase C/IP3 system, there are other ways by which an external stimulus can elevate intracellular calcium: • The NMDA receptor (a glutamate receptor in the brain is a ligand-gated calcium channel. • An activated G-protein opens a calcium channel. Example: A calcium channel in the myocardium is opened by the activated Gs -protein in response to epinephrine. • An extracellular messenger depolarizes the plasma membrane, and this opens voltagegated calcium channels. Examples: Neurotransmitter release and smooth muscle contraction. Voltage-gated calcium channels in smooth muscle cells can be blocked pharmacologically by calcium channel blockers.

276

Desensitization of Receptors

17.9

17.7. Cyclic GMP (cGMP). cGMP is formed by guanylate cyclase, an enzyme which occurs in both membrane-bound and soluble forms. cGMP is inactivated by various phosphodiesterases. It acts by activating protein kinases G. Second messenger functions: Atrial natriuretic factor (ANF) is a polypeptide hormone from the heart which causes natriuresis, vasodilation and a decrease of aldosterone synthesis. The ANF receptor has an intracellular guanylate cyclase domain that becomes active after ligand binding. Nitric Oxide (NO·), known as “endothelium-derived relaxing factor”, is formed in endothelial cells by an NO· synthase which uses arginine as a substrate. Vasodilators such as acetylcholine (muscarinic receptors), bradykinin, and histamine activate the NO· synthase through calcium-calmodulin. NO· diffuses from the endothelium to the vascular smooth muscle cells where it activates a soluble (cytoplasmic) from of guanylate cyclase, causing vasodilation. Nitrovasodilator drugs such as nitroglycerin are metabolized to nitric oxide. In retinal rod cells, visible light activates rhodopsin, a member of the 7-transmembrane receptor family. Rhodopsin is coupled to the G-protein transducin. The α-subunitGTP-complex of transducin activates a cGMP-specific phosphodiesterase: the extracellular stimulus (in this case not a hormone but light) decreases cellular cGMP levels. The decrease in cGMP hyperpolarizes the membrane.

17.8. Desensitization of Receptors Prolonged agonist exposure can desensitize receptors: Receptor phosphorylation: The agonist-induced conformation of the receptor may be sensitive to phosphorylation, which converts the receptor to a “useless” form. Example: Activated (but not inactive) β-adrenergic receptors become phosphorylated by β-adrenergic receptor kinase (BARK). The phosphorylated receptor can still bind epinephrine, but it can no longer interact with the Gs -protein. Also protein kinase A can phosphorylate and thereby inactive the β-adrenergic receptor. Receptor down-regulation: Agonist-stimulated receptors are physically removed from the cell surface by receptor-mediated endocytosis. Endocytosed receptors can be recycled to the cell surface, but some of them are degraded by lysosomal enzymes. The downregulation of insulin receptors is a mechanism of insulin resistance in type II (maturityonset) diabetes mellitus. Unlike receptor phosphorylation, down-regulation is longterm and can be overcome only by the synthesis of new receptor.

277

17.9

Biochemistry and Genetics

17.9. Objectives in Summary 1. Recognize the structural features of the major classes of steroid hormones and state the precursor relationships of these hormones. 2. Name the substrates and products of cytochrome P-450 dependent hydroxylation reactions. 3. Describe the sequence of steps in thyroid hormone biosynthesis and their cellular localization. 4. Describe the sequence of reactions, cofactors and precursor relationships in the biosynthesis of catecholamines, indolamines and histamine. 5. List the mechanisms for the inactivation of biogenic amines, including the mechanisms of the inactivating reactions and type of products formed. 6. List the typical steps in the processing of pro-hormone, using pro-insulin as an example. 7. Name the products that are formed in the cyclooxygenase and lipoxygenase pathways of eicosanoids metabolism. 8. Name the substrates for the synthesis of acetylcholine and GABA, the products formed in degradation of these neurotransmitters and the enzymes catalyzing these reactions. 9. Describe the typical kinetics of hormone receptors with respect to Bmax and KD . 10. Describe the mechanism of signal transduction by hormone-regulated G-proteins and name the second messengers that activate protein kinases A, C and G. 11. Outline the sequence of events in the phosphoinositide second messenger system. 12. Name the molecular targets of cholera toxin and pertussis toxin. 13. Describe the mechanism of action for steroid and thyroid hormones. 14. Name examples of agents that act through protein/tyrosine phosphorylation, by binding ligand-gated ion channels, or by inducing the synthesis or degradation of cGMP.

278

Part IV.

Semester two, Mini I

18. Vitamins and minerals This introduction will be necessarily brief. For more a more complete introduction to nutrition see [Shiles et al., 2005].

18.1. Nutrient doses All things are poison, and nothing is without poison. Only the dose makes that a thing is not poison. Philippus Theophrastus Aureolus Bombastus von Hohenheim (Paracelsus) “A lot helps a lot” is no proper approach to human nutrition. Substances which are required by the body in small amounts may be very toxic if given in too large a dose. For this reason experts in different countries have set up tables of dietary requirements. Most well known of these are the recommended dietary allowance (RDA) set by a group of nutrition scientists in the Food and Nutrition Board at the National Academy of Science of the USA, which are reviewed every 5 years. These represent the best judgement of a group of experts on the daily amounts of nutrients that are sufficient and safe for a healthy individual [Otten et al., 2006]. The RDA is calculated from the average requirement for a particular nutrient plus 2 standard deviations (see fig. 18.1). It therefore meets the requirements of about 98 % of the population. An average person requires 77 % of the RDA to maintain health. Failing to eat the RDA of a nutrient does not necessarily lead to deficiency, but the probability of a deficiency is the larger, the more uptake deviates from recommended values. In recent years, RDA-values were complemented by estimated average requirement (EAR) and tolerated upper intake level (UL)-values. Together with the RDA-value these form the Dietary reference intake (DRI). EAR is the dose required by an average member of the group studied. UL is that dose that will not cause adverse effects in the majority of subjects, even when taken regularly over many years. Note that if the EAR of a nutrient can not be determined it is also mathematically impossible to determine the RDA. In such cases adequate intake (AI) values are given instead of RDA-values. The AI-value is determined from the intake of a healthy reference population,

281

18.1

Biochemistry and Genetics

f(x)

F(x)

Nutrients: Beneficial and toxic effects

Dose

EAR RDA Estimated Recommended Dietary Average Requirement Allowance

UL Tolerated Upper Intake Level

LD 50 50% Lethal Dose

Dietary Reference Intake (DRI) If EAR has not been determined, RDA can not be set. In this case use AI (Adequate intake) instead of RDA.

Food and Nutrition Board of the National Academy of Sciences of the USA EB 2007

Figure 18.1.: Estimated Average Requirement (EAR): A nutrient intake value that is estimated to meet the requirement of half the healthy individuals in a group. Recommended Dietary Allowance (RDA): This value is a goal for individuals. It is the daily dietary intake level that is sufficient to meet the nutrient requirement of 97–98 % of all healthy individuals in a group. Tolerable Upper Intake Level (UL): The highest level of daily nutrient intake that is likely to pose no risks of adverse heath effects to almost all individuals in the general population. As intake increases above the UL, the risk of adverse effects increases. EAR, RDA and UL values together form the Dietary Reference intake (DRI). Somewhere beyond the UL lies the LD50 , the dose that would kill half of the studied population. LD50 is not part of the DRI, however. Note the break in the dose-axis.

282

Assessing nutrient stores and needs

18.1.2

hence it is an intake that by definition will cause neither deficiency nor toxicity in the majority of people. Remember: A deficiency can not be diagnosed from uptake data alone, but only from clinical, physical and biochemical data on a specific person.

18.1.1. The US Recommended Daily Allowance Recommendations for RDA are given separately for different genders and age groups, making the table a bit unhandy. For everyday purposes a simplified version of the recommendations exist, the US recommended daily allowance (US-RDA). It is important to clearly distinguish RDA and US-RDA, despite their similar abbreviations. The US-RDA is taken to be the highest RDA (based on 1968 recommendations) in any gender and age group above 4 years (in most cases the US-RDA is equivalent to the RDA of 18 year old males). Because US-RDA values are used for labeling of food packages and changes would be expensive to the industry, they are not updated like the RDA values, but have remained constant since 1968.

18.1.2. Assessing nutrient stores and needs The bodies pools of many micronutrients, in particular minerals and fat soluble vitamins, are difficult to determine. The nutrients are stored in tissues like bone or liver, and blood levels are fairly constant unless either the stores are so depleted that blood levels can no longer be maintained or the stores are so full that the excess can no longer be put in there (see fig. 18.2). RDAs for such nutrients are therefore difficult to determine. One lab technique used is to give a relatively large dose of the suspected nutrient. The amount excreted is then measured. If retention is high, body stores were depleted. Nutritional assessment is covered in [Gibson, 2005]. Additionally, needs for micronutrients, in particular ultratrace elements, may depend on physiological status, and may increase upon exposure to nutritional, metabolic, hormonal or psychological stress. In such a situation a subclinical deficiency may become manifest. For the ultratrace elements there is an additional problem: Very sensitive analytical techniques are required to determine their concentration in both food and tissue. Additionally, it is very difficult to exclude very small concentrations of trace elements not only from food and water, but also from cages, toys, scientific instruments etc., which might otherwise lead to contamination. The techniques continue to be developed, one reason why we expect to see further chemical elements declared essential in the future. To ensure adequate supply, trace elements and vitamins are sometimes added to processed food. We distinguish:

283

18.1.2

Biochemistry and Genetics

start of withdrawl

onset of symptoms

blood

stores

critical concentration

Figure 18.2.: Depletion of body stores (blue, dotted line) and blood concentrations (red, full line) of a nutrient over time. As the uptake of the nutrient ceases the body stores are depleted at a certain rate, but the blood concentration remains almost constant. Only after the body stores are so empty that blood concentration can no longer be maintained does it drop too. Symptoms of withdrawal appear when the blood concentration drops below a critical threshold.

284

Vitamins

18.2

Enrichment : when nutrients are added solely to replace losses during food processing Fortification : when extra nutrients are added to the food.

18.2. Vitamins Modern scientific understanding of nutrition started when Lavoisier identified the relationship between oxygen consumption and heat production. Liebig introduced the chemical analysis of food. He believed that carbohydrates and fats meet the bodies energy requirements, while proteins are used for growth. Dumas and Lunin found that a synthetic food composed of water, protein, carbohydrates and fat (the then known components of milk) does not support human growth: babied fed such a diet died. Funk proposed in 1912 that apart from these macronutrients small amounts of various substances are required, which he called vitamines (literally: nitrogen containing compounds necessary for life). The ‘e’ was dropped from vitamine in 1920, to reflect the fact that few of these compounds are amines, some do not even contain any nitrogen at all. Vitamins were originally divided in fat soluble vitamin A and water soluble vitamin B. In 1917 it became clear, that vitamin B contained a heat labile substance with activity against Beri-Beri (Vitamin B1 ) and a heat stable compound that promoted growth (Vitamin B2 ). 1925 Jansen & Donath isolated 5 g of pure vitamin B1 from 1 t of rice bran, the structure was finally solved in 1936, followed by the first synthesis. Today 300 t/a of synthetic Vitamin B1 are produced. In total 13 vitamins have since then been isolated, identified and synthesized, the last one was vitamin B12 in 1973. In this context it is important to remember that vitamins isolated from natural sources and those synthesized in the laboratory are identical substances and have, dose by dose, the same effects inside the body. There is therefore no need to use the (sometimes very expensive) “natural” vitamins for supplementation. Many “natural” vitamins even are synthetic products mixed into a natural base (like synthetic B vitamins mixed with yeast powder or synthetic ascorbate mixed with acerola). Vitamins are organic substances required by an organism in small amounts (µg/d or mg/d). They may act either as coenzymes or as participants in chemical reactions (for example antioxidants). Very high doses of vitamins may have additional, pharmacological or toxic effects. Research into such effects has only started. It is certainly unnecessary, but possibly also dangerous, to give vitamins at very high doses (exceeding 150 % US-RDA). “Megadoses” of more than 10 times US-RDA may be toxic even in the case of water soluble vitamins which can be excreted into urine. In case of fat soluble vitamins, which accumulate in the body, such doses are definitely dangerous.

285

18.2.1

Biochemistry and Genetics O

O O 2-Methyl-butadiene (Isoprene)

P O

O

P O

O

isopentenyl diphosphate (activated isoprene)

HO O HO 7-Dehydrocholesterol (Provitamin D)

Tocopherol (Vitamin E)

O OH

O Plastoquinone (Vitamin K1)

Retinol (Vitamin A1)

Figure 18.3.: Fat soluble vitamins are derived from isoprene (2-methyl-butadiene). The biological building block is isopentenyl-diphosphate (activated isoprene), which you have encountered in cholesterol synthesis. The building blocks are indicated by alternating blue and red segments. Vitamin E and K additionally contain aromatic residues produced in the shikimate-pathway, which is present in plants but not animals. Provitamins are compounds that can be converted into the active vitamin (for example carotene to vitamin A). Anti-vitamins (or vitamin antagonists) are compounds which are chemically similar to vitamins and can bind to their binding sites, without being able to fulfill their function. They are used as research tool, chemotherapeutic agents (e.g. methotrexate) or as antibiotics (e.g. sulphonamides). Vitamin deficiencies are still a major health problem in developing countries, but may be seen in the poor, homeless, drug addicts and psychiatric patients even in industrialized nations.

18.2.1. Fat soluble vitamins Fat soluble vitamins belong into the class of chemicals known as isoprenoids (isoprene = 2-methyl butadien, see fig. 18.3).

286

Fat soluble vitamins

18.2.1

Fat soluble vitamins are not excreted from the body, but accumulate in the liver if taken in excess. Thus most people have stores of these vitamins and they may be able to go without them for weeks or even months before deficiency becomes visible. On the other hand, if these vitamins are taken up in large excess over prolonged periods, toxic effects are seen. Fat soluble vitamins are absorbed in the small intestine dissolved in fat droplets also containing bile salts. Cave: Patients with reduced fat uptake (for example with intestinal or pancreas disease, cystic fibrosis, abetalipoproteinemia or on a low fat diet) may not receive enough fat-soluble vitamins! Retinal (Vitamin A) Function Vitamin A is required for vision (part of the “visual purple” rhodopsin in the light sensitive cells of the retina, which is bleached to “visual yellow” by light). However, only 0.01 % of vitamin A is found in the eyes, and 90 % is stored in the liver. The remainder is distributed throughout the body and serves to control reproduction and development: • Epithelial cells in mucous membranes require vitamin A. In its absence, keratinized cells without cilia are formed, which can not produce mucous. This may lead for example to respiratory infections. • Vitamin A controls the development of osteoblasts and osteoclasts and thereby bone formation. • Sperm production in males and maintenance of pregnancy in females depends on a sufficient supply of vitamin A. • Wound healing requires vitamin A, the mechanism is unclear. Vitamin A is actually a group of related compounds, called retinoids. Retinol, retinal and retinoic acid can be converted into each other by redox reactions, carotene consists of two vitamin A molecules linked tail to tail. Oxidation of retinal to retinoic acid is irreversible in the human body, and retinoic acid can not protect from the visual and reproductive effects of vitamin A deficiency. However, retinoic acid is an important morphogenetic hormone, which regulates growth and the formation of epithelia and bones. Until 1967 vitamin A amounts were measured by their biological effects in international units (IU). From then on “retinol equivalents” (RE) became the official unit of measurement. 1 RE is equivalent to 1 µg of retinol, 6 µg of β-carotene or 12 µg of other retinoids. This is equivalent to 3.3 IU of vitamin A. Clinically retinoic acid (not carotene) may be used for topical administration in acne patients (cave: may cause birth defects if given to pregnant females). β-carotene has been shown to interfere with proliferation and progression in certain cancers (especially lung cancer).

287

18.2.1

Biochemistry and Genetics

CH3 3

CH3

CH3

2

4

1 5

H3C

9

7 8

6

CH3

12

OH

15

13

11 10

14

CH3

Retinol

CH3

H3C

CH3

2 [H] H3C CH3

CH3

CH3

CH3

β-carotene

CH3

CH3

H3C

CH3 O

H3C

CH3

CH3

Retinal

CH3

H

11-cis-Retinal

H

[O] H3C

CH3

H3C

O CH3

CH3

CH3

O

Retinal reductase

H3C

CH3

Rhodopsin

Opsin

Retinol isomerase

OH

Light

Retinoic acid

CH3

CH3

CH3

H

O H3C

CH3

H all-trans-Retinal

Figure 18.4.: Different forms of vitamin A. The alcohol retinol is oxidized first to the aldehyde retinal (required for vision) and finally to retinoic acid, which is a hormone involved in morphogenesis. The conversion between retinal and retinol is fully reversible, but retinoic acid can not be converted into retinal by the human body. 11-cis-retinal is a component of rhodopsin (visual purple). Under the influence of light it isomerizes to all-trans-retinal (visual yellow), which dissociates from the protein component and is transported out of the retina for regeneration. 11-cis-retinal then returns to the cone cell stacks and recombines with opsin to form rhodopsin again. The light induced conversion of 11-cis into all-trans retinal also starts the signaling cascade in the photoreceptor cells.

288

Fat soluble vitamins

18.2.1

Food sources Yellow and orange plants, leafy vegetables, red palm oil, egg yolk, milk fat and liver provide high concentrations of vitamin A. Synthetic β-carotene is used as food color for example in lemonades. In developing countries with undersupply of vitamin A staple foods may be supplemented (cane sugar in central America, sodium glutamate in the Philippines). The use of red palm oil for cooking and the home growth of carotene rich vegetables should be encouraged. In some countries children < 5 a of age receive 30 000 RE of vitamin A in oil directly onto the tongue every 6 month. Uptake and metabolism Retinoic acid is readily absorbed in the intestine and transported in blood bound to albumin. Target cells bind it at a surface receptor, cellular retinoic acid binding protein (CRABP)). Retinol is usually taken up as retinyl palmityl ester which is split by pancreas juice. Retinol is absorbed by the intestinal epithelial cells with the aid of bile, uptake is vitamin E dependent. Inside the mucosal cell the palmityl ester is reformed and secreted with chylomicrons into the lymphatic system, from where it enters the blood stream and the liver, where it is stored in lipid droplets. Retinol is released (Zn2+ dependent) from the liver bound to retinol binding protein which binds to a receptor at the surface of the target cells, cellular retinol binding protein (CRBP). Carotene may be either split in the intestine by pancreas juice to retinol or taken up whole, this uptake (but not that of retinol) is regulated by vitamin A stores in the body. Carotene is stored in adipocytes and adrenals, not in the liver. Blood carotene concentration reflects food intake, not body stores. Excess uptake may cause a yellow tinge of the skin, carotenodermia. It may be converted in the body to retinol, but it may also be used as antioxidant. Excretion Retinoic acid can be excreted by the kidneys, and some carotene and retinol may be excreted in bile. Most retinol is stored in the liver however (about 150 000 RE in the average adult). Deficiency Night blindness, xerophthalmia (dry eyes), failure to produce tears, keratomalacia (excessive keratin in skin and conjunctiva), opaqueness and sloughing of the corneal epithelium (Bitot’s spots), rupture of the cornea, followed by infection and hemorrhage of the eye. Worldwide this leads to 250 000 cases of blindness per year, mostly in children. Many more may succumb before these symptoms develop.

289

18.2.1

Biochemistry and Genetics

Figure 18.5.: Bitot’s spot, eye lid changes and cataract in a patient with vitamin A deficiency. Hypovitaminosis A is still the worlds most common cause of preventable blindness, even though it can be prevented for a few cents per patient and year. Image from webeye.ophth.uiowa.edu/.

290

Fat soluble vitamins

18.2.1

Other signs of vitamin A deficiency include sensitivity to infections, developmental problems, sterility, a rough, dry skin (especially around the shoulders), folliculosis (permanent goose bumps), acne. Changes in the gastrointestinal epithelia may lead to diarrhea. Tooth enamel may fail to form, sense of taste and smell may be lost. Toxicity High doses of vitamin A (more than 20 000 RE/d) given to pregnant female may cause birth defects in the infant, for example cleft palate. Vitamin A poisoning has been observed after a diet high in liver (historically seen in polar exploration). Symptoms include headaches, drowsiness, nausea, loss of hair, dry skin, diarrhea, anorexia, skeletal pain, resorption of bone, cessation of menstruation in females, beginning several months after the start of high uptake. Infants are more sensitive, symptoms may appear after 1 month and may also include hydrocephalus, increased intracranial pressure and hyperirritability. Because the uptake of carotene is regulated, plant sources of vitamin A are safe. Genetic diseases relating to vitamin A metabolism The regeneration of all-trans- to 11cis-retinal can not be done in the retinal rods. Instead, the all-trans-retinal is transported to the blood stream by a specific ABC-type membrane ATPase, ABC-R. Mutations in this transporter lead to accumulation of all-trans retinal and its metabolites in the cone cell, which become poisoned. In the mildest cases (miss-sense mutations) this leads to Stargardt-disease. More severely affected patients suffer from autosomal macular dystrophy, in the worst cases (frame shift or splicing mutants with total loss of ABC-R function) retinitis pigmentosa results. In autosomal macular dystrophy the extend of damage also depends on environmental factors, which are not fully understood yet. Calciferol (Vitamin D) Function Calcitriole, the active form of vitamin D, is produced in the kidney under the influence of parathormone and stimulates the synthesis of a Ca2+ and Pi binding protein in the gut and thereby the absorption of these nutrients from food. Calcitriol also stimulates Ca2+ and Pi resorption in the kidney and Ca2+ release from the bones. Amounts of vitamin D in the food are measured in international units (IU), 1 µg = 40 IU. Because the synthesis of cholecalciferol in the skin requires UV light, nutritional needs for vitamin D are particularly high in people whose occupation or clothing habits exclude them from sunlight.

291

18.2.1

Biochemistry and Genetics

26 25

18

19

CH3

2

3

HO

4

5

10

11

12

13 14

9 8 6 7

27

23

21

24 20

22

17 16 15

7-dehydro cholesterole

h*ν

UV-light (skin)

CH2

Ergocalciferole

identical!

Vitamin D2, from yeast + plants

H2C

HO

Cholecalciferole (Calciole, Vitamin D3)

OH

Liver

OH

CH2 Calcitonine

HO

25-Hydroxy cholecalciferole

(Calcidiole) Ca, Pi

Kidney Parathormone

OH

OH OH

Calcitonine

OH CH2

(OH) CH2

Kidney 1,25-Dihydroxy cholecalciferole

HO

(Calcitriole, active forme of vitamine D)

HO

24-hydroxylated vitamin D (inactive, excreted)

Figure 18.6.: Metabolism of vitamin D. 7-dehydro cholesterol can be produced in the human liver. Opening the ring system to form cholecalciferol (vitamin D3 ) is possible in the human skin only if it is exposed to UV light. Cholecalciferol is converted to 25-hydroxy cholecalciferol (calcidiol) in the liver, which is then converted (under the control of parathormone) in the kidney to the active form of vitamin D (Calcitriol, 1,25-dihydroxy cholecalciferol). This substance controls the uptake and metabolism of Ca2+ and P. Ergocalciferol (vitamin D2 ) is a compound found in yeasts, which can substitute for cholecalciferol. It is used as a vitamin D supplement.

292

Fat soluble vitamins

18.2.1

Figure 18.7.: Genu varum (bend femurs) and reduced bone density in a 2-year old child with rickets. Image from en.wikipedia.org.

Pregnant woman also have high needs for vitamin D, because about 1/2 of the fetal Ca2+ is deposited in the last 6 weeks of pregnancy. Needs are also high during lactation, as breast milk contains large amounts of Ca2+ . Food sources liver, fish, yeast, milk (cave: patients with lactose intolerance!). Uptake and metabolism Vitamin D is absorbed in the upper part of the small intestine with 80 % efficiency, transported in chylomicrons to the blood stream, where it is attached to α-globulin2 (vitamin D binding protein) and transported to the liver, together with the vitamin D produced in skin. In the liver it is converted to calcidiol, transported to the kidney and converted to calcitriol, the active form. The later reaction is controlled by parathormone under the influence of low serum [Ca2+ ]. High serum [Ca2+ ] leads to the release of calcitonin from the thyroid gland, which stimulates the hydroxylation of calcidiol and calcitriol in the 24-position, leading to inactive products (see figure 18.6) . Patients receiving anticonvulsants or tranquillisers may have larger needs for vitamin D. Excretion Some vitamin D may be excreted in bile and feces.

293

18.2.1

Biochemistry and Genetics

Deficiency If not enough vitamin D is available, Ca2+ and Pi metabolism are dysregulated. This leads to a low muscle tonus and to weak bones and teeth, which are unable to fulfill their function. In children this is called rickets, in adults osteomalacia. Patients suffering from rickets are easy to recognize, the leg bones bow under the weight of the body (O-legs), the rib cage is concave and narrow and crowds the internal organs (pigeon breast). The fontanel fails to close (should normally happen by 1 year of age), teeth erupt late, are ill formed and decay rapidly. Toxicity In sensitive children doses as low as 400 IU/d may lead to hypercalcemia, nausea, weight loss and failure to grow. Calcidiol increases the production of the interleukin IL-2 in T-lymphocytes. That interferes with the maturation of TH1 - and dendritic cells, resulting in a TH2 -answer and IgE production against environmental antigens. Thus the risk for allergies is increased, indeed, according to some authors the increase in the morbidity from allergies is at leat in part due to increased vitamin D supplementation in children. During pregnancy a hypervitaminosis D may lead to impaired maturation of osteoblasts and defective bone formation in the embryo.

Tocopherol (Vitamin E) Function Tocopherols and tocotrienols include a large number of related compounds, of these α-tocopherol is most active. The activity is measured in tocopherol equivalents (TE), 1 TE = 1 mg of RRR-α-tocopherol = 1.49 IU. Synthetic tocopherol supplements contain an inactive stereoisomer, hence their activity is, weight for weight, only half of the natural product. Vitamin E is an antioxidant and radical scavenger. It prevents oxidation of polyunsaturated fatty acids in cell membranes, in particular of the red blood cells and in the lungs. In this role it partially overlaps with the function of Se. Needs for vitamin E increase with the uptake of poly-unsaturated fatty acids (PUFA), but fortunately the best sources of PUFA also contain a high concentration of vitamin E. Other functions for vitamin E in cell signalling, which have been described in the literature, are now understood as consequence of the protection of PUFA, which in turn have signalling function, e.g. arachidonic acid. (Trabera & Atkinson, 2007). Vitamin E will protect vitamins C and A in food against oxidation during storage. Clinical uses of vitamin E: intermittent claudication (cramps in calf muscle) and fibrocystic breast disease (painful but harmless knots).

294

18.2.1

Fat soluble vitamins

CH3 OH H3C CH3

O CH3

α-tocopherol

CH3 OH

this compound has the highest vitamin E effect

H3C CH3

O CH3

α-tocopherol

CH3

R

OH H3C

RH

CH3

O

O

CH3

free radical R

+

H2O

RH CH3

H3C

O H3C

CH3

O γ-tocopherol

CH3

O

OH

glutathione ascorbate

H3C

CH3

β-tocopherol

CH3

O OH quinone

CH3 CH3

Figure 18.8.: Left: Tocopherols include a large number of different compounds (which might be isolated for example from wheat germ oil), but only tocopherol α, β and γ have vitamin E activity. Right: Oxidation of α-tocopherol by free radicals. You have seen a similar stepwise reaction with ubiquinone (CoQ) in oxidative phosphorylation.

295

18.2.1

Biochemistry and Genetics

Food sources Plant oils, nuts, wheat germ, asparagus, avocado, mango, spinach Uptake and metabolism Vitamin E is absorbed with fat in the upper part of the small intestine and transported in chylomicrons and LDL. It rapidly exchanges with cell membranes. Patients suffering from cystic fibrosis have low fat absorption capacity and may require supplementation with 50–400 TE/d. Excretion Vitamin E is stored in adipocytes, muscle and liver, so little excretion occurs. Deficiency flaky skin, weak muscle (nutritional muscular dystrophy, muscle fiber fragmentation), liver degeneration, loss of membrane function, hemolysis (resulting in anemia), increased lipofuscin formation. Hypovitaminosis E may also be involved in cancer and sterility (tocos (gr) = birth). However, a recent study (SELECT) on protection against prostate cancer had to be aborted as no beneficial effect could be demonstrated either alone or in combination with Se. Similarly, no beneficial effect against breast and lung cancer has been detected in other studies. In premature infants low stores of vitamin E combined with a limited ability to absorb fat may lead to increased sensitivity to oxygen damage, in particular to the retina (retrolental fibroplasia). 100 TE/d α-tocopherol protect, if given either by injection or orally in a water miscible form. Toxicity Hypervitaminosis E is rare. Symptoms include gastrointestinal distress, nausea, diarrhea and failure of blood clotting (made worse if vitamin D is low). Epidemiological studies have shown increased all-cause mortality if intake exceeds 2000 mg/d, an UL of 1000 mg of α-tocopherol was established. Vitamin E related inherited diseases In ataxia and vitamin E deficiency (AVED) the inability of the liver to pass vitamin E into the blood stream leads to severe neurological defects. Patients require supplementation with an injected, water-miscible vitamin E preparation. Vitamin K Function Vitamin K is a group of compounds required for blood coagulation (spelled with ‘k’ in many languages other than English). The need for this compound was discovered by Danish Carl Peter Henrik Dam in chicken 1929, the substance was characterized by American Edward Adelbert Doisy. Both shared the 1943 Nobel price for Medicine.

296

18.2.1

Fat soluble vitamins

Glu

Gla

O 2,4-Naphtoquinone

CO Prot phytyl

O

3

CH3 O

Phylloquinone (plants, vitamin K1)

OH

O

Prot

N CH H CH2

isoprenyl-

OH

O

n=4-13

Menaquinone (bacteria, vitamin K2)

O

O

N CH H CH2

C HOOC H COOH CO2

H

O

+

CH3 O

OH

R OH

CH3

Prot

C HOOC H H

O

Warfarin

CH3 O

CO Prot

O

Phenprocoumon (Marcoumar)

H2O

Vit. K (hydroquinone) Warfarin 2 [H] Vit. K epoxide reductase

OH

O2

γ-glutamyl carboxylase

R O Vit. K (epoxide) 2 [H]

O CH3

CH3

Vit. K epoxide reductase

R O O Menadione (synthetic, vitamin K3)

Coumarin

O

O Vit. K (quinone)

Figure 18.9.: Left: Different forms of vitamin K: Phylloquinone is found in plants, menaquinone in meats. Menadione is a synthetic form of vitamin K. Note that in the older literature quinones are spelled (etymologically correct!) chinons. Middle: Vitamin K antagonists used as anti-coagulants (“blood thinner”) and rat poison. Right: Conversion of Glu to Gla by γ-glutamyl carboxylase, thus creating a Ca2+ binding site. The enzyme requires vitamin K in its reduced form (hydroquinone) as cosubstrate, the vitamin is oxidized to the epoxide in the process. The epoxide is returned to the active form in a 2-step process, both steps are catalyzed by vit. K epoxide reductase. This enzyme is inhibited by vit. K antagonists.

297

18.2.1

Biochemistry and Genetics

The mode of action of this vitamin however was discovered only in 1974 by several independent groups. Vitamin K is a cofactor in the synthesis (in the liver) of factors II (prothrombin), VII (proconvertin), IX (thromboplastin) and X (Stuart-factor) of the blood clotting cascade. For activity, these factors require the conversion of a glutamic acid residue to γ-carboxyglutamic acid (abbreviated Gla). The change from Glu to Gla creates a Ca2+ binding site in the protein. Osteocalcin, a Ca2+ binding protein in bone and possibly other proteins require Gla formation as well, as does Gas6, a anti-apoptotic factor in cells. Conotoxins (from a marine snail, Conus geographus) and some snake venoms also contain Gla. Bacteria like E. coli use menaquinone as an intermediary for anaerobic respiration, where electrons are transferred from lactate or NADH to other electron acceptors than oxygen, e.g. nitrate forming nitrite. Two naturally occurring compounds have vitamin K activity, phylloquinone (vitamin K1 ) in plants and menaquinone (vitamin K2 ) in animals and bacteria. Menadione is a synthetic compound (vitamin K3 ), which is activated to vit. K2 in the body. Like with other synthetic K-vitamins it’s use in animal feeds is now frowned upon, it should not be used in humans. Food sources About half of our supply of vitamin K is produced by our intestinal flora. Salicylates, sulphonamides and antibiotics reduce intestinal synthesis. Vitamin K is also contained in leafy green vegetables (1 Tbs of parsley = 150 % RDA) and in smaller concentration in fruits, tubers, seeds, eggs, dairy products and meats. Cooking can reduce vit. K content of food, steaming is recommended instead. Uptake and metabolism Vitamin K uptake in the small intestine is aided by bile and pancreas juice. Obstruction of the bile duct and oil based laxatives interfere with vitamin K uptake. Efficiency of uptake is about 10 %. Body stores of vitamin K are small and turnover is rapid. Bacterially produced vitamin K in the end gut is absorbed passively with relatively low efficiency, even this uptake however requires bile acids. Excretion Vitamin K may be stored in the body for about 1–2 wk maximum in liver and adipose tisue, metabolites are excreted into the bile and to some extend also into urine. Deficiency Deficiency is usually the result of malabsorption and leads to hemorrhaging, stomach pain, bone malformation (especially in children) and deposition of Ca2+ -salts in arteries. Since such deposits are part of atherosclerosis it has been speculated that vitamin K RDA may be too low.

298

18.2.2

Water soluble vitamins

CH3 NH2 N H 3C

H C C O H2 H2

N + N

O

S

CH3

NH2

O N

P O P O O

Thiaminediphosphate

O

H3 C

OH

HC C C O H2 H2

N + N

O

S

CH3

O

P O P O O

O

Hydroxyethyl-thiamine diphosphate

(activated vitamin B1)

(activated acetaldehyde)

Figure 18.10.: Thiamin diphosphate, the metabolically active form of thiamin, serves as a cofactor for dehydrogenases and transketolases. Toxicity has been observed only after massive overdose of synthetic vitamin K derivatives: Hemolytic anemia, increased blood bilirubin (jaundice) followed by bilirubin accumulation in the grey matter of the brain. This then leads to mental retardation. Vitamin K1 and K2 are considered non-toxic, a UL has not been set. Suspicions that injected vitamin K could lead to leukemia have not been confirmed in later studies. Vitamin K related genetic diseases Newborns are at risk for late-onset hemorrhagic disease of the newborn (HDN) after about day 8, for this reason in many countries a prophylactic vitamin K dose is given. Since the condition is rare the benefit is questionable, however, it will do no harm.

18.2.2. Water soluble vitamins The capacity of our body to store water soluble vitamins is much smaller than for the fat soluble. Thus overdoses of water soluble vitamins tend to be less toxic, because the excess is excreted from the body, on the other hand, vitamin deficiencies develop much faster as there are no body stores to draw on. Thiamin (Vitamin B1 ) Function Thiamin diphosphate, the metabolically active form of thiamin, serves as cofactor for dehydrogenases and transketolases, for example: • oxidative decarboxylation (pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched chain α-ketoacid dehydrogenase) where the remaining molecule is transferred to CoA. • transketolase (e.g. in pentose phosphate pathway)

299

18.2.2

Biochemistry and Genetics

• Conversion of tryptophan to niacin • Neurotransmitter release for high frequency impulses, the mechanism is unknown • the metabolism of ethanol and acetaldehyde It is thought that Beri-beri is caused by the accumulation of toxic intermediates from inhibition of transketolase. Clinically thiamin is used to reduce nausea in pregnancy. Food sources meat, fish, poultry, legumes, nuts, whole grain cereals, less in milk and vegetables. Thiamin deficiency is usually associated with the change from unprocessed to processed grain products, for example polished rice or white flour. Thiamin (and some other nutrients) are contained mostly in the aleuron layer of the grain, which is removed during processing. Thiamin is sensitive to heat, oxygen, and alkali (baking soda). Some foods contain a thiaminase, which destroys the thiamin. In fish and seafood, this enzyme is heat labile, but black tea contains a heat stable form. Uptake and metabolism Thiamin is taken up in the duodenum by active, Na+ coupled transport after the hydrolysis of any phosphate groups by intestinal phosphatases. Sulfonamides and some antibiotics interfere with uptake. The human body contains about 30–70 mg of thiamin, half of that is found in muscle. Excretion in urine. Deficiency Hypovitaminosis B1 leads to Beri-beri, a life threatening disease. Beri-beri is still a major public health problem in some developing countries. About 1.5 × 106 people worldwide are said to suffer from thiamin deficiency of various degrees. For example in the Philippines there are 75 infant deaths per 100 000 births linked to Beri-beri, it is the fourth leading cause of death. Beri-beri is characterized by lack of motoneuron coordination (Beri-beri literally means “I can’t, I can’t”), weakness, wasting of muscles, un-coordinated movements, convulsions, confusion, apathy, high heart rate (tachycardia), heart hypertrophy, edema, high serum pyruvate, cyanosis, vomiting. Babies have a thin, almost inaudible cry. Cause of death is usually heart failure.

300

Water soluble vitamins

18.2.2

Onset is most frequently in infants of 2–5 months, very rapid and requires treatment within hours to prevent death. Cause is usually a thiamin deficiency in the mother, who then produces a milk with low thiamin content, which also contains pyruvic aldehyde, a toxic metabolite. In adults there are two forms of Beri-beri: - wet (edematous) with fluid accumulation starting in the feet and moving upwards. Death occurs from heart failure as the chest becomes congested with liquid. - dry (wasting) with gradual loss of body tissue and emaciation. Further symptoms in both forms include numbness of the legs, irritability, disorderly thinking, constipation (because of low muscle tone in the GI tract), nausea, anorexia. Alcoholics, whose “liquid nutrition” does not contain vitamin B1 , who may suffer from intestinal degeneration (limiting uptake) and who have a high need for this vitamin to detoxify the ethanol, may suffer from Wernicke-Korsakoff-disease. Wernicke’s encephalopathy is an acute CNS dysfunction (delirium) and can be cured with thiamin. Korsakoff’s psychosis (anterograde amnesia with confabulation) is irreversible and requires institutionalization. Toxicity none known for uptakes up to 100 mg/d per os. Injected thiamin seems more dangerous and should be avoided.

Riboflavin (Vitamin B2 ) Function Riboflavin in the form of FAD and FMN is a cofactor of oxidoreductases (see fig. 18.11). Food sources Milk, egg, whole grain cereals, yellow vegetables, liver, kidney. Riboflavin is stable against heat, acid and oxygen, but destroyed by alkali and exposure to light. Uptake and metabolism Uptake of riboflavin in the upper part of the small intestine is regulated by thyroid hormone. Uptake efficiency is about 70 % if riboflavin is taken with a meal, but only 15 % if taken separately. Riboflavin is converted to FMN in the intestinal epithelial cells and transported in blood to the liver bound to albumin. There it is converted to FAD.

301

18.2.2

Biochemistry and Genetics Riboflavin (Vitamin B2)

O

H N

H3 C H3 C

N

N

H O

Riboflavin (Vitamin B2)

N

H 3C

N

H 3C H

N

N

N

CH2

H

HC OH

HC OH HC OH

HC OH

HC OH

CH2

CH2

OH

OH

H

H3 C

N

O

H 3C

N

CH2

H

N N

HC OH HC OH

HC OH

HC OH

O

Flavin mononucleotide (FMN)

O

H 2C

H2 N N

HC OH

O

O

H

CH2

HC OH

O P O

H

O

H N

N

O

HC OH

O

H H 3C

H N

H 3C

2 [H]

CH2

H

H

H N

O

N N

N OH O O

OH

O P O P O O

O

Flavine adenine dinucleotide (FAD)

Figure 18.11.: Riboflavin, in the form of the coenzymes FMN and FAD is a component of oxidoreductases. Excretion Riboflavin is excreted in urine, after the kidneys have reabsorbed the bodies needs. Small amounts are excreted in bile.

Deficiency psychological deviations (hypochondriasis, depression, hysteria), cheilosis (cracked and inflamed lips), glossitis (smooth, purple-red tongue), growth retardation, reduced hand grip strength, fatty dermatitis, anemia. A lack of vitamin B2 during embryonal development leads to malformations (cleft palate, cataracts, shortening of the long bones and fusion of ribs). Cave: Since riboflavine is light sensitive it gets destroyed during phototherapy of newborns suffering from hyperbilirubinemia. Supplementation is required in such cases.

Toxicity No riboflavin toxicity has been observed up to 20 mg/d, which is the limit of uptake capacity.

302

18.2.2

Water soluble vitamins

H3 C O

O

C O

C

N

NH2 N

N

Nicotinic acid

N

Nicotin (not a vitamin, toxin in tobaco)

Nicotinamid O C

O

O

N

NH2

O C

+

H N NH2

O P O O

HO

N

OH

O P O

N O

O

OR

HO

NH2

N N

Isoniacid (tuberculostatic drug)

N

R = H: NAD+ R = Pi: NADP+ H H H

O C

N

+

H

R

NAD(P)+

NH2

2 [H]

H H

H

H

N

O C

NH2

H

+

H

+

R

NAD(P)H + H+

Figure 18.12.: Nicotinamide is a component of NAD+ and NADP+ , which are used as cofactors in dehydrogenases to catalyze the transfer of reduction equivalents. Nicotine is not a vitamin, but a toxin from tobacco. In the American literature nicotinic acid and nicotinamide are called niacin, to prevent the wrong impression that tobacco consumption would supply a vitamin. Isoniazid is an antivitamin used as tuberculostatic drug.

303

18.2.2

Biochemistry and Genetics

Nicotinamide and nicotinic acid (Niacin, Vitamin B3 ) Function Nicotinamide is a component of NAD+ and NADP+ , which are cofactors in dehydrogenases. Nicotinamide can be synthesized in the human body from tryptophane, 60 mg of Trp are equivalent to 1 mg of nicotinamide (1 niacin equivalent NE). Proteins on average contain about 1 % Trp, thus 6 g of protein are equivalent to 1 NE. Conversion of Trp to NAD(P) requires thiamin, pyridoxin, riboflavin and biotin. Cave: In ~ estrogen inhibits the conversion of Trp to niacin, making them more susceptible to pellagra than |. Food sources Meat, legumes, nuts, fish. Corn contains nicotinamide in a chemically bound, unavailable form. Treatment of corn with dilute bases releases it (nixtamalization). South American Indians traditionally treat corn with a suspension of burned lime Ca(OH)2 to achieve this effect. Niacin is chemically stable under conditions normally employed in food preparation. Uptake and metabolism Vitamin B3 is absorbed in stomach and upper part of the small intestine. Excretion in urine as methyl derivative. Deficiency Lack of nicotinamide, leads to pellagra (Ital. pelle = skin; agra = rough) characterized by “the four D’s”: diarrhea, dermatitis, dementia and death. Black tongue disease in dogs is used as an animal model for pellagra. Treatment is by oral vitamin B complex or yeast, this may be accompanied with a tranquilizer in patients with psychiatric symptoms. Recovery should occur within a few days. A diet high in Leu and low in Ile may also cause pellagra, this can occur in areas where sorghum or millet is a staple food. Isoniazid (INH, isonicotinic acid hydrazide) is a tuberculostatic drug which acts as vitamin antagonist. Pellagra may occur in patients treated with this drug. Alcoholics have high needs for vitamin B3 for the detoxification of ethanol. Corn was introduced in the 17th century into southern and eastern Europe and widely planted because of its high yields. However, the habit of nixtamalization was not transferred from the new world. This led to a pellagra epidemics in Europe and later in the southern US, especially in spring when little other food was available. First description of pellagra was in 1735 by Gaspar Casal, Goldberger in the 1930’s recognized pellagra as

304

Water soluble vitamins

18.2.2

Figure 18.13.: Dermatitis on sun-exposed body parts in pellagra. Image from Ashourian & Mousdicas, NEJM 354 (2006) 1614.

305

18.2.2

Biochemistry and Genetics

a vitamin deficiency. The myth of vampires is claimed to originate from these epidemics: The light-sensitive dermatitis made patients avoid daylight. After several episodes of erythema, the patient’s skin becomes parchment-like. Bleeding sores in mouth and tongue make blood run down out of the victims mouth, the glossitis makes the tongue appear red. Brain-damage results in dementia and a manic-depressive state, making the behavior of the victims unpredictable and potentially dangerous. Pica leads to the patient eating life animals (zoophagia: flies, spiders, birds). Mucus membrane lesions lead to the patients refusal to eat solid food. The high number of deaths from an unknown, chronic wasting disease was attributed to a nosferato (from Gr. plaque carrier) seeking revenge on his/her neighbors and family after death. Toxicity Nicotinic acid, but not nicotinamide, is a vasodilator. Doses in excess of 50 mg lead to flushed skin, tachycardia, hypoglycemia and burning or tingling sensations. Excessive supplementation with vitamin B3 leads to gastrointestinal distress, nervousness, recurring ulcers, glucose intolerance and fulminant hepatitis. Under strict medical supervision, 1–2 g/d may be used to treat high blood cholesterol and schizophrenia (“orthomolecular therapy”), higher doses have no beneficial effect, but single doses of up to 9 g do not seem to cause acute toxicity. Pantothenic acid (Vitamin B5 ) Function Pantothenic acid is a component of Coenzyme A, which is required as acyl carrier. The energy-rich thioester bond preserves the energy of C−C-bonds split in making the acyl-CoA. Pantothenic acid is clinically used to stimulate gastrointestinal mobility after surgery. Pantothenol is used in cosmetics, its mechanism of action (if any) is unclear. Food sources all living matter (pan (Gr) = everywhere). Particularly rich are organ meats, fish, whole grain cereals and royal jelly. Pantothenic acid is destroyed by dry heat. Uptake and metabolism Excretion Out of an average uptake of 13–19 mg/d 2–7 mg appear in urine and 1–2 mg in feces.

306

18.2.2

Water soluble vitamins

O

OH

OH

N

O

CH3 CH3 OH

Pantothenic acid

Adenine Pantothenic acid Mercaptoethyl amine

Ribose-3'-phosphate

O

O HS

N

N

CH3

O

O

O P O P O CH3 OH

Coenzyme A

O

N O

O

O

N

N OH

N

N

O P O O

Figure 18.14.: Pantothenic acid is a component of Coenzyme A, which is used as acyl-group carrier in biochemical reactions (Acetyl-, succinyl-, propionyl-, fatty acid and HMG-CoA). The thioester bond to the acyl group is energy rich. Deficiency Lack of pantothenic acid causes unspecific symptoms resulting from reduced cell health, including a higher rate of infection, fatigue, insomnia, vomiting. Reports that pantothenic acid could prevent the greying of hair have not been substantiated.

Toxicity Very large doses (more than 10 g) cause diarrhea.

Pyridoxine (Vitamin B6 ) Function Pyridoxalphosphate is a cofactor in transaminase, deaminase and decarboxylase reactions. These occur in energy metabolism, amino acid metabolism, hormone synthesis (serotonin, histamine, taurine, GABA), cross-linking of elastin, synthesis of RNA and heme, synthesis of arachidonic acid from linoleic acid, cholesterol synthesis and turnover, synthesis of nicotinamide. The requirements of vitamin B6 depend on the protein intake, 16 µg are needed per g of protein.

307

18.2.2

Biochemistry and Genetics H2C OH HO

HC O HO

CH2OH

H3 C

H 3C

N

H2C NH2 HO

CH2OH N

H 3C

Pyridoxal

Pyridoxin

HC O HO

O H2 C O P O

CH2OH N

Pyridoxamin

H 2C

NH2 O H2 C O P O

HO

O H 3C

N

+ HO

Pyridoxal phosphate (bound to transaminase)

C

O H3 C

N Pyridoxamine phosphate (bound to transaminase)

+ O

H2 N C H R aminoacid

HO

C

O

O C R keto acid

Figure 18.15.: Pyridoxalphosphate is a cofactor in transaminase, deaminase and decarboxylase reactions. Pyridoxin, pyridoxal and pyridoxamine can all be used by the human body to produce pyridoxalphosphate. Food sources Pyridoxin is widely distributed, white meats, liver, whole grain cereals, egg yolk, bananas, avocados, green leafy vegetables and potatoes are good sources. Pyridoxin is sensitive to alkali, light and oxygen. Uptake and metabolism Absorption is by passive diffusion in the jejunum with 40–80 % efficiency depending on the food source. Pyridoxin is transported in blood bound to albumin. Absorption of pyridoxin is inhibited by more than 40 drugs, including oral contraceptives (add 10 mg/d of pyridoxin to the requirements), isoniazid, penicillamine. Excretion In urine mostly as pyridoxic acid, in smaller amounts also as pyridoxamine, pyridoxal and pyridoxal phosphate. Deficiency Weakness, difficulty in walking, microcytic hypochromic anemia, insomnia, neuromotor seizures, hyperexcitability, dermatitis. In developed countries vitamin B6 deficiency is mostly seen in alcoholics.

308

Water soluble vitamins

18.2.2

Low uptake leads to increased oxalate in urine, this may lead to urinary calculi. If deficiency occurs in the first few months of life, permanent brain damage may occur. Infants suffering from hypovitaminosis B6 give a characteristic, piercing cry. During pregnancy a lack of pyridoxin increases the risk of toxemia, a fatal complication. Women who took oral contraceptives before becoming pregnant may enter pregnancy with low stores of pyridoxin. Supplementation with pyridoxin may help to control nausea. Large doses (up to 200 mg/d) are used for the symptomatic treatment of carpal tunnel syndrome. Toxicity Doses of more than 2 g/d lead to loss of motor coordination, which is reversible upon withdrawal. Regular supplementation with more than 200 mg/d may lead to dependency. Cobalamine (Vitamin B12 ) Function Vitamin B12 is required for • Several mutases in which a hydrogen and some group on an adjacent carbon exchange places. This may be followed by an elimination of water or ammonia. • Methyl group transfers and recovery of folate. • Other reactions in microorganisms and plants. Food sources Bacteria (cyanocobalamine), dairy products (methyl- and hydroxocobalamin) and fish, poultry and meat (adenosylcobalamine). Contrary to public opinion intestinal bacteria can not supply the body with cobalamine, as they occur in the large intestine, where little if any uptake occurs. On the contrary in veterinary medicine antibiotics like aureomycin and penicillin are given to speed weight gain in cows in part by killing cobalamine destroying bacteria in their gastrointestinal system (intestinal physiology and anatomy is quite different in ruminants and man). Uptake and metabolism Vitamin B12 is in the stomach bound to “intrinsic factor”, a heat labile glycoprotein produced in the parietal cells of the stomach. The complex of vitamin and intrinsic factor is then absorbed in the small intestine, the reaction depends on Ca2+ . Uptake efficiency is 1–60 % depending on supply. In blood cobalamine is transported bound to transcobalamine (a protein) to reduce renal loss. Cobalamine is stored in the liver, most people have stores for several years.

309

18.2.2

Biochemistry and Genetics

R= CN: Cyanocobalamine (Vitamin B 12)

O

C

R= 5'-desoxyadenosine: Adenosylcobalamine (Coenzyme B 12) R= CH3: Methylcobalamine (cofactor for C1-transfer)

H2 N

HO

N

N

N N

H2N

OH

O

R

O H2N

5'-desoxyadenosine

CH2

H3C

O C H2 H3C C H2 C

CH2

O C C H2 H2

3+

Co

CH3 N

H2 C H2 C

N

CH3CH3

C NH2

CH3

N N

C C H2

NH2

CH3 CH3

C C H2 H2N O

C NH2 O

CH2 CH2 HN

C O

CH2

N N

H3C C O P O H O OH

H2C

CH3

O CH3

O

OH

O odd chain fatty acids

C

S CoA

HC CH2 O C H O Methylmalonyl-CoA

B12 MethylmalonylCoA-mutase

O

C

S CoA

HC CH2 H C O O Succinyl-CoA

Figure 18.16.: Cobalamine has a heme like structure with a central Co atom. Various ligands can occupy the 6th position of the Co coordination sphere. One reaction which requires cobalamine is the exchange of a hydrogen atom against a neighboring group, for example in methylmalonyl-CoA mutase. If methylmalonyl-CoA is not eliminated by this reaction, it’s concentration will increase until it is utilized instead of malonyl-CoA for the synthesis of fatty acids. The resulting branched chain fatty acids get incorporated into phospholipids and destabilize myelin sheaths.

310

Water soluble vitamins

18.2.2

Excretion with urine, pancreas juice and bile.

Deficiency Lack of vitamin B12 leads to pernicious anemia, where red blood cell precursors can not divide for lack of DNA. This leads to fewer, very large (megaloblastic) erythrocytes. Because of failure in fatty acid biosynthesis in the brain patients also show demyelinization. Pernicious anemia is usually the result of failure to produce intrinsic factor, so that uptake efficiency is reduced. The condition was originally treated by giving the patient 300 g/d raw liver pulp (Whipple 1920 followed up by Minot & Murphy. All three shared the Nobel Price for Physiology and Medicine in 1934). Liver contains high concentrations of cobalamine and the high dose overcomes the uptake deficiency. Life quality for the patients was improved as more and more concentrated extracts could be prepared, which could be injected i.m. (Cohn 1938). Since 1973 vitamin B12 can be made synthetically, intrinsic factor from hog stomach is now also available. Apart from genetically determined inability to secrete intrinsic factor, a cobalamine deficiency may be caused by: - a strict vegan (as opposed to vegetarian) diet - removal of stomach - defective mucosal lining of the stomach, caused by genetic problems, Fe-deficiency, alcoholism, thyroid dysfunction - tapeworm infections - lack of transcobalamine, the transport protein in blood - therapy with anticonvulsants or certain antibiotics - reduction of gastric acid production in old age, needed to liberate cobalamine from protein complexes in food - Inherited lack of uptake protein in the intestine (Schilling-disease) - Autoantibodies against transport protein in blood or cell surface receptor

Toxicity No toxic reactions have been observed so far even in relatively large doses, because amounts exceeding the binding capacity of transcobalamin are rapidly excreted by the kidneys. Allergies have been reported though.

311

18.2.2

Biochemistry and Genetics

O C

R O

HO C

R OH

O

HO C

O C

.O

R O

C

R OH

O

HO C

H2O

OH

O C HC OH HO CH

C OH H2

Ascorbic acid radical

C

O C

alkali, light, heat

HO CH

C OH H2

L-Ascorbic acid

O

HC

HO CH

C OH H2

O C O C

HC

HC HO CH

O

O C

C OH H2

L-Dehydroascorbic acid

Diketogulonic acid physiologically inactive

HO

C

O

HO

CH2

CH2 CH2

C

O2

CO2

O

H

H H H

N

O

Gly

Peptidyl-Proline

similar: Hydroxylysine

+

H

X

OH NH2 O H2N C C C C C C H2 H H2 H2 H OH

OH

Succinate Proline hydroxylase

+ H H

C

O

α-ketoglutarate

H

O

CH2 O C HO

C

H

HO H H

H H N X

O

Gly

Peptidyl-4-Hydroxyproline

Figure 18.17.: L-Ascorbic acid (Vitamin C) is required for the hydroxylation of proline and lysine in connective tissue and acts as an antioxidant. Under the influence of light and heat diketogulonic acid is formed, this compound has no vitamin C activity.

312

Water soluble vitamins

18.2.2

L-Ascorbic acid (Vitamin C) Function Only L-Ascorbic acid has vitamin C function, the D-form is used under the name of erythroic acid as a meat preservative. Ascorbic acid is required for - the introduction of hydroxy groups into lysine and proline residues in collagen, which are required for the correct folding of this extracellular matrix protein. The reaction is Peptidyl-Pro + α-ketoglutarate + O2 → Peptidyl-Hydroxyproline + succinate + CO2 . The hydroxylase requires Fe2+ (ferrous) in its catalytic center. Sometimes succinate is formed without hydroxylation of proline or lysine. In this case Fe is oxidized to the 3+ (ferric) form, and requires reduction by ascorbic acid to regenerate the enzyme (α-ketoglutarate + ascorbate + O2 → succinate + dehydroascorbate + CO2 + H2 O). A similar mechanism probably explains the requirement for ascorbate in the synthesis of tyrosine, carnitine, hydroxymethyluracil, uracil and uridine. - formation of norepinephrine from tyrosine and serotonin from tryptophan - fatty acid desaturation (regeneration of cytochrome b5 ) - C-terminal amidation of neuropeptides with glycine at the C-terminus (for example αmelanotropine): Peptidyl −CO−NH−CH2 COOH + O2 + 2H+ → Peptidyl−CO−NH2 + H2 O + OCH−COOH. This reaction is catalyzed by an ascorbate- and Cu2+ -dependent monooxygenase. - Val and Ile synthesis (not in humans) - conversion of cholesterol to bile salts - reducing dietary Fe3+ to Fe2+ for uptake - chelating minerals like Fe2+ , Zn2+ , Ca2+ for uptake - protecting dietary vitamins like folic acid from oxidation - detoxification in the liver - histamine detoxification (allergies) - sulfate metabolism: formation of ascorbic acid sulfate Dehydroascorbic acid is regenerated using glutathione. Pharmacological doses of vitamin C (more than 1 g/d) are sometimes recommended to protect against cancer and colds. Both efficacy and safety of such doses have not been established.

313

18.2.2

Biochemistry and Genetics

Food sources After Lind showed in 1747 that juice from citrus fruits could cure scurvy, it became law that any ship leaving an English harbor had to carry sufficient lime juice for the journey, hence the nickname “limey” for British service men. Ripe fruits and vegetables contain considerable amounts of vitamin C. Of meats, only liver is a good source. Cows milk contains little vitamin C, and most of that is destroyed during sterilization. Infants raised on cows milk therefore require supplementation to avoid the rapid onset of lethal scurvy. Most infant formulas take care of this however. Breast milk contains sufficient vitamin C for the infants needs, provided the supply for the mother is high enough. Ascorbic acid is frequently added to processed food as antioxidant to stabilize color and flavor, protect sensitive nutrients like PUFA in margarine and to maintain an acidic pH.

Uptake and metabolism Ascorbic acid is absorbed in the upper part of the small intestine both by diffusion and by Na+ -dependent uptake. Uptake efficiency depends on the dose. The maximal serum concentration of 1.2 mg/l is reached at a dose of 45 mg/d, this is equivalent to total body stores of 1500 mg. If uptake of vitamin C stops, body stores will decrease by about 3 % per day, scurvy will start at 300 mg total body stores. Smoking reduces vitamin C absorption, effectively doubling the required dose.

Excretion Vitamin C is excreted into urine, reabsorption is regulated to maintain the serum level at 1.2–1.5 mg/l.

Deficiency Lack of vitamin C leads to scurvy. Patients become listless, tired, suffer from body aches, muscle cramps and loss of appetite. The joints swell. The skin becomes dry, rough and feverish. Because of the weak connective tissue small hemorrhages lead to purple spots (petechia) on the skin. The gums will also be affected, teeth will fall out and secondary infections cause additional damage.

Toxicity At doses in excess of 500 mg/d ascorbic acid is converted to oxalic acid, this may lead to kidney stones in the 20 % of the population genetically predisposed to this disease. Megadoses in pregnant woman may lead to dependency in the infants. There is also evidence for oxidative damage caused by excess uptake of ascorbic acid (production of superoxide radicals by the Fenton-reaction).

314

18.2.2

Water soluble vitamins

Figure 18.18.: Scurvy resulting from vitamin C deficiency. As Pro and Lys in collagen can no longer be hydroxylated, connective tissue is weakened leading to petechia. Pictures from phil.cdc.gov.

O HN

O N H

H

HN

H

C CH2 H2C H S CH2

CH2

CH2 H N H C R

O

CH2 C N H H

CH2 HCO3

O

CH2 CH2

CH2

Biotin

CH2 HN

H

C CH2 H2C H S

CH2

C

N COO-

H

HN

-

H2O

O

R

O O

O

CH2 CH2

Lysin residue

H N HC

C

CH2 H N H C R

O

CH2 C N H H

H N HC

O

O O

R

Protein chain

Biocytin

Carboxybiocytin

Figure 18.19.: Biotin is required as cofactor for carboxylases, to which the biotin is chemically bound at a lysin residue.

315

18.2.2

Biochemistry and Genetics

Biotin Function Biotin dependent carboxylases are required for synthesis of fatty acids (AcetylCoA carboxylase for the production of malonyl-CoA), glucose (pyruvate carboxylase for the synthesis of oxaloacetate), nicotinic acid, purines and prostaglandins. The metabolism of Val, Ile, Met and Thr requires propionyl-CoA carboxylase and the metabolism of Leu β-methylcrotonyl-CoA carboxylase. Of these enzymes acetyl-CoA carboxylase is cytosolic, the remaining enzymes occur in mitochondria. Biotin is also required for the formation of antibodies and pancreatic amylase. Food sources Widely distributed. Biotin from animal sources tends to be protein bound and fat soluble, plant biotin free and water soluble. Rich sources are liver, kidney, egg yolk, yeast, legumes (in particular sprouted), nuts, cauliflower and whole grains. Biotin is heat stable, but is unstable under alkaline conditions and may be oxidized. Uptake and metabolism Biotin bound to lysine (biocytin) is released from proteins by proteolytic digestion. Biocytin is hydrolyzed by biotinidase and taken up in the jejunum by active, saturatable transport and by passive diffusion. Anticonvulsants interfere with uptake. Biotin is transferred to a lysine group in the apo-carboxylases by holocarboxylase synthase in a ‘ping-pong’ reaction: Biotin and ATP bind to the synthase and bound biotinyl-5’-AMP is formed under release of pyrophosphate. Then the apo-carboxylase binds to the synthase, and biotin is transferred to it. Finally, the holoenzyme and AMP are released. Excretion in urine, in part as metabolites. Deficiency Maculosquamous dermatitis, alopecia, low appetite, nausea, depression, seizures, encephalopathy, glossitis, immune suppression. A deficiency may occur after the ingestion of a large number of raw eggs, because the egg white contains a factor (avidin) which binds biotin with very high affinity (Kd = 1 × 10−15 M). Toxicity none known.

316

Water soluble vitamins

18.2.2

Inherited diseases relating to biotin Neonatal multiple carboxylase deficiency (neonatal MCD) is caused by a holocarboxylase synthase which has a reduced affinity for biotin. The disease becomes apparent soon after birth. Late onset MCD becomes apparent when the infants are weaned, that is when they switch from free biotin in mothers milk to biocytin in normal food. It is caused by biotinidase deficiency. In both cases supplementation with very high doses of biotin (up to 10 mg/d) can bring relief.

Folic acid (Vitamin M, Vitamin B9 ) Function Folic acid is a C1 -carrier, this is discussed in detail in the chapter on amino acid metabolism.

Food sources Wheat germ, liver, kidney, yeast, mushrooms, fruits and leafy vegetables (folio (Lat.) = leaf). Folate is destroyed by heat, light and oxidation.

Uptake and metabolism After extraneous Glu residues have been split of, folate is absorbed with 50–90 % efficiency in the upper part of the small intestine. Uptake is vitamin B12 dependent. Folate is then transported by the portal blood to the liver and from there to the rest of the body. Inside the cells of the body it is conjugated with a chain of up to 12 Glu molecules to prevent it from leaving the cells. In the liver folate is stored as methyl folate, release requires vitamin B12 . Normal liver stores are about 7–15 mg, which is sufficient for almost half a year. Oral contraceptives, antitumor agents, anticonvulsants and excessive ethanol consumption interfere with folate uptake. Tropical sprue will also lead to uptake deficiency.

Excretion because folate is converted to the polyglutamate inside the cells, very little is excreted.

317

18.3

Biochemistry and Genetics

Figure 18.20.: Megaloblastic anemia. Images curtesy of Dr. http://www.va.gov/telepathvisn6/Hematpth.htm.

W.M.

Todd,

Deficiency megaloblastic anemia, weak immune system. During pregnancy higher risk of complications like hypertension and damage to the embryo: neural tube defects and “small for date” births. Therefore reasonable folate supplementation during pregnancy is recommended. Folate deficiency is considered to be the worlds most prevalent vitamin deficiency, in particular in children, pregnant females and old people. Toxicity Folate in very high doses interferes with the uptake of Zn2+ and may promote the growth of certain tumors. Folate can to some extent relieve the megaloblastic anemia and glossitis seen in pernicious anemia. However, it exacerbates the neurological problems and makes the disease more difficult to diagnose from blood smears. For this reason many countries limit the folate content of vitamin supplements.

18.3. Minerals A large number of elements have been shown to be essential for human nutrition, some of which were thought of as contaminants only a few years ago. We distinguish

318

Mass elements

18.3.1

mass elements needed in amounts of more than 100mg/d. These include Ca, Mg, Na, K, Cl and P. They are present in the body in concentrations of more than 50 ppm. trace elements needed in amounts greater than 1 mg/d. These include Fe, Zn, Cu and Mn. ultratrace elements needed in amounts of less than 1 mg/d. These include As, B, F, I, Se, Cr, Co, Mo, Si, V, Ni, Sn. Several other elements may also be required (suspected are Ba, Sr, Cd, Br). Traces of most other elements are found in the body too, but it is unknown whether or not they have any function. Trace element concentration in food stuff is determined by soil concentration. Modern international marketing of food, where food comes from many different regions, has made deficiencies in trace elements much rarer. Milk and dairy products contain little trace elements, this is also true for human milk. Infants should therefore receive supplementation with other food from 4–5 m of age onwards, when their body stores – acquired during pregnancy – become depleted. Both required amounts of micronutrients and their toxicity in high doses have to be considered. Most micronutrients can be obtained in sufficient amounts from food, supplementation should be considered for Ca, Fe and I only, unless very unusual circumstances dictate otherwise. This issue is made even more complicated by the fact that there are synergistic and competitive interactions between minerals (Se − Hg, Ca − Pb, Fe − Zn, Mo − Cu, Mo − S) and between minerals and vitamins (Ca - vitamin B12 , Se - vitamin E).

18.3.1. Mass elements Ca2+ and P These elements are conveniently considered together, not only because they are both required for the formation of bone and teeth, but also because their uptake is influenced by the presence of the respective other element. Function About 1.5–2 % of body weight is Ca2+ (about 1 kg) and about 1 % is phosphorus. 90 % of both elements are found in bones and teeth in the form of hydroxyapatite (a crystalline material containing Ca(OH)2 , Ca3 (PO4 )2 and CaF2 in variable amounts, with Mg2+ , Zn2+ , Na+ , CO2− 3 also present). The crystals are incorporated into a protein matrix (collagen in bones and keratin in teeth). Ca2+ is also found in the blood and soft tissues (≈ 10 g), where it has several functions:

319

18.3.1

Biochemistry and Genetics

• Blood clotting: When platelets are injured, Ca2+ influx releases thromboplastin from their cell membranes. This in turn stimulates the conversion of prothrombin into thrombin. • Stimulation of the absorption of vitamin B12 and some other nutrients. • As intracellular messenger involved for example in the regulation of muscle contraction and insulin secretion • Cofactor of enzymes like acetylcholine esterase and lipase. An antioxidative enzyme in skin is Ca2+ dependent, Ca2+ thus is useful in treating burns and slowing the aging of skin. • Ca2+ lowers blood pressure and may to some extend counteract high blood pressure caused by excessive Na+ . It also lowers cholesterol levels. • Ca2+ stabilizes DNA and RNA structure. • Ca2+ is used for the prevention and treatment of arthritis, rheumatism, menopause problems, menstrual cramps and nephritis, although the mechanism isn’t quite clear. Ca2+ is a natural tranquilizer and required for good sleep. When your grandma recommended you take a glass of warm milk with honey before you go to sleep, she may not have known what she was doing, but her advice was certainly sound. Phosphorus too has many functions in the body apart from tooth and bone formation: • Energy metabolism • Phospholipid formation • Phosphorylation for signaling and transport • DNA and RNA formation • Component of cofactors (eg thiaminphosphate) and enzymes • Regulation of blood pH

Food sources Ca2+ and Pi are contained in milk and dairy products, grains, fish, meat and some vegetables. Phosphate is also contained in some processed food like soft drinks and sausages.

320

18.3.1

Mass elements

Calcidiole

g/l 0m 7 <

Parathyroid

carrier-dependent uptake in small intestine

Parathormone citrate release from osteoclasts

Blood [Ca]

Calcitriole >7 0m

g/l

parafolicular cells (C-cells) of thyroid

Calcitonine

Ca-retention in kidney 24-hydroxylated metabolites Osteoblast function

Figure 18.21.: The calcium concentration in blood is closely regulated. Uptake and metabolism Ca2+ is taken up with 10–30 % efficiency (in adults, children up to 75 %) by active transport into the epithelial cells of the small intestine, particularly the uppermost part, were the chyme is still slightly acidic. Oxalate, phytate, fatty acids, stress and lack of exercise (cave: bedridden patients!) interfere with Ca2+ absorption, as do factors that increase the transport speed of food through the intestine. Caffeine, mineralocorticoids, thyroxin and anticonvulsants also interfere. Lactose and glucose increase Ca2+ absorption, as does a food Ca2+ /Pi ratio between 1:2 and 2:1. Phosphate is released from food materials by phosphatases in the digestive juices. Its uptake in the small intestine, resorption in kidney and bone metabolism is controlled by parathormone and calcitonin in much the same way as that of Ca2+ . Al(OH)3 based antacids interfere with Pi absorption by forming insoluble AlPO4 . Maintaining Ca2+ and Pi in a soluble form The concentration of Ca2+ (1.2 mM) and Pi (1.3 mM) in blood serum is higher than their solubility product (nominal solubility product of [Ca5 (PO4 )3 (OH)] is 10 × 10−53 M), i.e. these ions should precipitate in the form of hydroxyapatite. Such precipitation of course would be fatal. Several factors are responsible for maintaining these ions in a soluble state: factors affecting the solubility product: The presence of NaCl and other salts in the blood increases the solubility product. Also, at the pH of blood most of the phosphate is not PO3− 4 .

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low molecular weight molecules forming complexes with Ca2+ : The most important one is pyrophosphate. Bone mineralization requires the activity of pyrophosphatases, which make the Ca2+ available and also increase the local concentration of phosphate. proteins forming complexes with Ca2+ : Many proteins – including serum albumin – can bind Ca2+ with moderate affinity on acidic amino acid side chains. Thus the concentration of free Ca2+ is reduced. colloid stabilization: Certain proteins like fetuin A bind to small (50–100 nm) hydroxyapatite particles and prevent them from precipitation. The colloid is removed from the blood stream for example by phagocytes. Thus humans avoid “Lot’s wife’s problem” 1 (W. Neumann). However, unwanted calcification is involved in the pathomechanism of some diseases, most notably atherosclerosis.

Excretion Ca2+ is lost from the body in urine (100–175 mg/d), feces from gastrointestinal secretions (130 mg/d) and sweat (20 mg/d).

Deficiency Low Ca2+ and Pi lead to bone demineralization, osteoporosis and osteomalacia. Low blood [Ca2+ ] additionally leads to tetany from higher nerve excitability and high blood pressure. Low blood [Pi ] leads to fatigue and loss of appetite. Soy has been shown in studies to reduce Ca-loss in females after menopause.

Toxicity High blood [Ca2+ ] leads to the contraction of muscle fibres (Ca-rigor). There is circumstantial evidence linking excessive Ca2+ -uptake to prostate and ovarian cancer. A high uptake of Pi from processed foods (soft drinks, sausages etc.) has been linked to attention deficit disorder in children.

Na+ and K+ Again it is easier to treat these two ions together. Both are important electrolytes, Na+ occurring mainly in the extracellular, K+ mostly in the intracellular fluid. This imbalance is maintained at the expense of metabolic energy by the Na+ /K+ -ATPase (Na+ -Pump) in the cell membrane, which pumps 3 Na+ out of and 2 K+ into the cell for each molecule of ATP hydrolyzed. This results in a ratio Na+ /K+ of 1:10 inside and 28:1 outside the cell. 1

In Gen. 1915−26 Lot’s wife was turned into a salt pilar because she looked back onto Sodom and Gomorrah while they were destroyed for their sins.

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Function Na+ maintains the osmotic pressure of blood and extracellular fluid. The concentration gradient of Na+ across the cell membrane is used to power secondary active transport processes for many nutrients (for example glucose and amino acids). By neutralizing acids Na+ is involved in the regulation of blood pH. K+ in the same way is involved in the regulation of the intracellular pH and osmotic pressure. Some enzymes like the 70 kDa 70 kDa heat shock cognate (Hsc70) (“uncoating ATPase”) require K+ as cofactor. The disequilibrium of both Na+ and K+ results in an electrical potential across the cell membrane. This allows signal conduction in nerves as well as the excitation of muscle and gland cells. Food sources Fruits, vegetables and meat contain large amounts of K+ . In the form of alginate, iodate and nitrate K+ is used in food processing. Beware however of K+ leaching into the cooking water. Na+ is widely available in our food in the form of table salt, usual uptake of NaCl is about 7–18 g/d. Only a small part of this is “discretionary salt” (salt which we add our self and can therefore control). In addition, Na+ as bicarbonate, glutamate, citrate, phosphate, saccharinate, benzoate, sorbate, propionate and nitrite is widely used in food processing, these compounds account for ≈ 10 % of daily Na+ intake. Uptake and metabolism Na+ and K+ are absorbed mostly in the small intestine. Excretion About 90–99 % of the Na+ ingested is excreted by the kidneys, depending on the amount taken up in food. This is regulated by aldosterone. A high blood [Na+ ] will activate thirst receptors, to ensure a sufficient water supply for excretion. 200 mg/l Na+ is found in sweat. Deficiency About 500–700 mg/d of Na+ would be adequate for the bodies needs, however a diet with only this amount would be unpalatable. Additionally, some Na+ is stored in the body bound to bone to cover a short lack of supply. However, large losses with sweat or diarrhea must be replaced, to prevent water intoxication. K+ too is usually supplied in sufficient quantities in the food. However, increased losses occur in diarrhea, vomiting and after the use of diuretics. If not enough carbohydrate is taken up with the diet, the body has to use protein instead to supply glucose for brain and erythrocyte function. This leads to a dangerously high loss of water (to excrete urea) and therewith also electrolytes. This can lead to circulatory failure.

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Toxicity About 20 % of the US population suffer from Na+ -dependent hypertension, which is caused or at least worsened by high dietary Na+ uptake. Hint: Replace part of the discretionary salt with herbs and spices, and add 1/4 of those only in the last 10 min of cooking. It also helps to keep a ratio of about 1:1 between dietary Na+ and K+ . Outright Na+ -poisoning may occur when people drink sea water, for example after a ship wreck . Sea water contains more Na+ than can be excreted with the water, thus increasing thirst. Victims then drink more sea water until death occurs from kidney failure. High blood [K+ ] leads to lack of muscular coordination, tissue breakdown and acidosis. Eventually death occurs by kidney and heart failure. In some countries that still have capital punishment injection of KCl solution is used for execution (death by lethal injection).

Chloride Function Chloride is an important counter-ion for Na+ and K+ . By neutralizing bases formed in the body it participates in the regulation of pH. This is particularly true in erythrocytes, to compensate for the constant change between the formation and removal of bicarbonate (chloride shift). High concentrations of chloride are found in gastric secretions and in cerebrospinal fluid. Chloride is found mostly in the extracellular fluid, to balance the negatively charged proteins inside the cells. About 0.15 % of body weight is Cl− . Food sources Chloride is widely distributed in food, and added in the form of NaCl. Excretion Excretion is by the kidneys, were reabsorption occurs when supplies are low. Deficiency In one case several children died after being fed an infant formula from which the chloride had been left out. Like Na+ and K+ , Cl− needs to be replaced after losses by diarrhea, vomiting, diuresis and excessive sweating.

Mg2+ Function Mg2+ is involved in more than 300 known enzyme reactions, amongst them those that require nucleotides or phosphate. Mg2+ is a Ca2+ antagonist in nerve activity. About 30 g are found in an adult body, 60 % of this in bone (some as magnesium phosphate in the bone structure, the rest loosely bound as Mg2+ store). Mg2+ is 7 times more concentrated in the intracellular than the extracellular fluid.

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Food sources Green plants contain high amounts of Mg2+ , but some is found in most other food as well. Water may also supply some Mg2+ . However, the Mg2+ supply in the average diet, at least in the US, is marginal. Uptake and metabolism Mg2+ is absorbed in the small intestine with 35–40 % efficiency. Ca2+ , ethanol, Pi , phytate and fat decrease, vitamin D and lactate increase absorption efficiency. Absorption and excretion of Mg2+ are regulated by the thyroid and parathyroid glands in much the same way as Ca2+ . Excretion In the kidney. Deficiency Mg2+ deficiency may be caused by malnutrition, vomiting, diarrhea, surgical trauma and high Ca2+ intake. Ethanol and diuretics increase Mg2+ loss. Consequences are irritability, nervousness, vasodilatation, muscle cramps and convulsion (Mg2+ tetany). In extreme cases heart failure occurs. There seems to be a connection between soft tissue calcification and low Mg2+ intake. Mineral losses with sweat in endurance sports may lead to muscle cramps, for which lack of Mg2+ is largely responsible. Toxicity High Mg2+ concentrations have an anaesthetic effect, eventually leading to coma and death by heart failure. Clinically, this can result from kidney failure, when Mg2+ excretion is repressed.

18.3.2. Trace elements Iron An adult human body contains about 3 g of iron, about 70 % of this as hemoglobin, 7 % in iron containing enzymes and 4 % in myoglobin. Most of the rest is stored in the liver. As about 1 % of all red blood cells are turned over each day, 25 mg/d of iron are metabolized. Most of this iron is recycled, only about 1 mg/d are lost (shed intestinal cells, urine, skin, hair). Loss can triple in menstruating females (usual loss during menstruation is about 35 ml blood, equivalent to 18 mg iron. Blood loss may rise to 200 ml/cycle in clinical cases). Pregnancy and lactation also place increased demands on iron supplies. Hookworm infections lead to a loss of about 0.2 ml blood per day and worm, this can amount to a blood loss of 200 ml/d in heavy infections. About 500 × 106 people world-wide

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are infected with this parasite. Infections with Giardia can also cause iron deficiency. Both parasites may cause deficiencies in other nutrients as well. Food sources Iron is contained in red meat, liver, eggs and many vegetables. Only a small part of the iron contained in food is actually resorbed. Resorption is prevented by complex forming agents particularly in plant foods, like oxalic, tannic and phytic acid. Iron in human breast milk (but not cows milk) can be resorbed to about 50 %, iron from heme in red meat to about 30 %; there is apparently a special transporter for heme-iron in the gut. Iron in many plants is resorbed only with about 10 % efficiency, in rice only 1 %. Free iron can be absorbed only in the ferrous (Fe2+ ), not in the ferric (Fe3+ ) form. Reducing agents like ascorbate (Vitamin C) therefore increase iron resorption. Iron is most soluble (and easiest resorbed) in an acidic environment. Usual eating habits tend to satisfy caloric needs of the body, with the need of micronutrients being satisfied as a side effect. Women have on the one hand a higher dietary need of iron, but at the same time a lower need for energy (lower basal metabolism, smaller body size). These factors conspire to make the risk of iron deficiency higher in women (probable Feintake 9 mg/d) than in men (probable iron intake 11 mg/d). On the other hand, men (and postmenopausal women) are more at risk of iron overload. Uptake Food iron is complexed with gastroferrin when it arrives in the stomach. Hereditary lack of gastroferrin leads to impaired iron resorption. Actual resorption takes place in the small intestine (duodenum and jejunum). In the gut cells iron is complexed with ‘protein C’. It is than transferred either to transferrin for transport to the liver or to ‘protein S’ for short term storage in the gut cell. Iron binding to transferrin is dependent on the iron concentration in blood and therefore iron demand of the body. Excess iron is initially stored with protein S, from where it can be efficiently transferred to transferrin. In case of large excess iron is transferred from protein S to ferritin, from which it will not be mobilized again: As gut epithelial cell have a live span of only a few days this iron is lost eventually with the stool. Transferrin (siderophilin in older literature) is a soluble glycoprotein. It is the only known asymmetric single polypeptide with two binding sites for the same substrate. The binding sites are assumed to be equivalent, without co-operativity between them. The iron-transferrin complex (holotransferrin) is bound at the cell membrane of target cells at a specific receptor. This transferrin receptor has at neutral pH a high affinity for holo-, but a low affinity for apotransferrin. Receptors with bound transferrin are preferentially sorted into clathrin coated pits and endocytosed via clathrin coated vesicles. The vesicles with the receptor/holotransferrin complex fuses with the endosome, where the complex is exposed

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Trace elements

Compounds forming Fe complexes NH2 CH2 CH2

OH OH

HO

CH2 CH2 CH2

O C

HO N

O

C O

CH2 OH

CH2 CH2 C O NH CH2

H HO

O

O

HC OH

C H

H C O

C H

O C

OH OH

C O

CH2 CH2 CH2 CH2

OH

HO OH

HO N C O

an example for tannic acids

CH2 CH2 C O NH CH2 CH2 CH2 CH2

~O Pi ~ O Pi ~ O Pi

O ~ Pi O~ Pi

O ~ Pi

Myoinosit hexaphosphate (Phytate)

CH2 HO N C O CH3 Deferoxamine

HO O

C C

O OH

Oxalic acid

Figure 18.22.: Tannic acid, phytate and oxalate give insoluble precipitates with iron and interfere with its uptake (and that of other bivalent metals). Deferoxamine is used for chelation therapy.

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cell membrane

Sorting Binding

fusion

dissociation

coated pit

Vesicle formation

coated vesicle budding uncoating and fusion

Endosome (pH ~5)

Figure 18.23.: The transferrin cycle. Iron (red) loaded transferrin (green) binds to the transferrin receptor (purple) at the cell surface, which is then internalized by clathrin (blue) coated vesicles. In the low pH environment of the endosome the iron dissociates, but conformational changes in both apo-transferrin and receptor ensure that these proteins stay together for recycling to the cell membrane. At the cell surface the apo-transferrin/receptor complex is exposed to neutral pH and apo-transferrin dissociates. This frees the receptor for a new cycle. Figure taken from [Buxbaum, 2007].

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to low pH (about 5.5). Under these conditions transferrin rapidly looses the bound iron, but remains bound to the receptor because at this low pH the receptor has a high affinity for unloaded transferrin. The transferrin/receptor complex returns to the cell membrane in recycling vesicles. At the cell surface the transferrin/receptor complex is again exposed to neutral pH, where the affinity of the receptor for apotransferrin is drastically reduced. The complex therefore dissociates and apotransferrin is released to the blood stream. The ferric iron is reduced inside the endosomal system to ferrous iron by an unknown electron donor (NADH, ascorbate, glutathione?) and then passes the endosomal membrane into the cytoplasm. Blood contains haptoglobin and hemopexin to scavenge any hemoglobin or heme respectively resulting from hemolysis (about 10 % of red blood cells lyse in the blood stream instead of being taken up by macrophages). The resulting complexes are then taken up by hepatocytes for iron recycling. Only part of the transferrin molecules in blood are loaded with iron. Total Iron Binding Capacity (TIBC, in µM) is the concentration of bound iron plus the free capacity of the serum to bind iron (measured by adding a known amount of iron and determining the amount not bound by transferrin). If the iron concentration is divided by this value, the relative iron saturation is obtained. Example: TIBC = 56 µM, serum iron concentration = 43 µM. This yields a saturation of 100 % * 43 µM / 56 µM = 77 %. Normal iron saturation is about 30 %. Iron bound to transferrin must be in the ferric (Fe3+ ) state, while gut cells can only resorb ferrous iron (Fe2+ ). Oxidation from the ferrous to the ferric state is achieved by Ferroxidase I and II. Ferroxidase I is also known as ceruloplasmin, and also acts as a copper transporting protein. Iron supply in the cell is regulated by the rate of synthesis of transferrin receptor and apoferritin. In the case of transferrin receptor, the breakdown of mRNA is regulated, in case of apoferritin the rate of translation. Both mRNAs contain iron responsive elements (hairpins of 30–40 nucleotides) outside their coding sequence to afford iron dependent regulation. Storage Ferritin is a 24 subunit protein made up of 2 types of polypeptides: The H chain has a molecular mass of 21 kDa, the L chain of 19 kDa. The two forms of apoferritin have different tissue distribution, the heart contains mainly ferritin H, the liver and spleen mainly ferritin L. Ferritin can bind 20 % of its weight in iron, 2500 ions per ferritin molecule. Iron is bound as a mixed hydroxide, phosphate and oxide complex. Iron enters the complex as Fe2+ , and is than oxidized to the 3+ state by an unknown mechanism. A small proportion of the ferritin leaves the cells, again by unknown mechanisms, and shows up in serum. Serum ferritin levels mirror the body’s iron supply. However, some disorders increase serum ferritin levels, thus covering an iron deficiency.

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Figure 18.24.: Left: Prussian blue/safranine O staining for iron deposits (here in dermatitis). Picture taken from Rivera, Ishihara & Mihara, Arch. Dermatol. Res. 295 (2003) 19. Middle: Blood smear from a normal and right: from a dog suffering from iron deficiency. Note the irregularly shaped, irregularly sized erythrocytes (microcytic) with large central pallor (hypochromic). Image from Woods, Tarpley, Johnson & Latimer, http://www.vet.uga.edu/vpp/clerk/mwoods/.

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If a cell contains more iron than it can store bound to ferritin, it stores the remainder as hemosiderin. Hemosiderin is a iron oxide aggregate with organic components. It occurs in lysosomes, and increased hemosiderin content may eventually damage the lysosomes. In histological sections hemosiderin is stained by the Prussian Blue reaction. Deficiency If the body lacks iron, it can no longer produce hemoglobin in sufficient amounts. It will produce fewer, smaller red blood cells which contain less hemoglobin than usual (microcytic hypochromic anemia). This will impair oxygen transport. Patients will feel exhausted and short of breath. They will be of pale (or even bluish) skin color. Some circumstantial evidence links iron deficiency with immuno-suppression and increased infections. Serum ferritin levels below 12 µg/ml or iron saturation below 16 % indicate iron deficiency. Treatment is by iron supplementation, usually in the form of ferrous sulphate or gluconate (in children 6 mg/(kg day), adults 50 mg three times a day), and ideally combined with ascorbic acid. Supplementation requires regular laboratory controls. If the patient lives in an area where parasites such as hookworms or Giardia are endemic (or has visited such an area), the presence of those parasite should be investigated. Other causes for blood loss (for example intestinal cancer) also need to be excluded. Toxicity Too much iron is toxic to the body, causing hemochromatosis. A normal human liver contains about 1 g of iron, this can increase to 40–50 g in severe cases of hemochromatosis. Serum ferritin levels can reach 6000 µg/l (normal 20–250 µg/l). Causes for hemochromatosis are - congenital failure to regulate apoferritin synthesis. This creates an iron sink in the tissue, leading to lower iron saturation and increased iron uptake. - congenital excess iron absorption. The condition has been linked to chromosome 6 and occurs most often in patients with HLA-A3. This may specifically affect the uptake of heme iron. - excess iron intake. This can occur if rusty cooking utensils are routinely used, if iron supplements are taken in excess (poisoning with those interesting looking green iron pills accounts for 1/5 of all poisoning cases in children in the US → keep all medicines out of reach of children!) - alcoholism. Alcoholic drinks like red wine contain high concentrations of iron, additionally alcohol affects iron uptake regulation. - liver cirrhosis or portacaval shunt - pancreatitis. Pancreas juice is involved in iron uptake regulation.

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- professional exposure to iron, for example in miners - reduced use of supplied iron, for example in thalassemias. Frequent blood transfusions may add to the problem (250 mg Fe in 500 ml blood). In iron overload, the iron is deposited throughout the body as hemosiderin precipitate. This causes damage to the cells. Particularly affected are liver, pancreas and muscle. Symptoms of hemochromatosis include: - Chondritis and arthritis, in particular in fingers and hand. Initial symptom in about half the patients. - Increased melanin production in the skin - Hypopituitarism, leading to dwarfism and sexual infantilism - Gonadal atrophy (particularly in |) - Cardiomyopathy, arrhythmias and heart failure - Splenomegaly - Addison’s disease (adrenal gland) - Fibrosis and islet cell destruction, leading to Diabetes mellitus. Together with the darker skin this leads to the syndrome of bronze diabetes. - Liver cirrhosis, liver failure, portal hypertension and hepatocellular carcinoma Treatment is by blood letting (phlebotomy) (500 ml/week over 2 a). Chelation therapy with deferoxamine (an iron chelator isolated from Streptomyces ssp.) and vitamin C is usually not recommended because of side effects. Fluorine Function Replacement of hydroxy-groups in hydroxyapatite in teeth by F− makes them harder and more resistant to tooth decay. In bones, this reaction makes the hydroxyapatite more resistant to demineralization. Thus a good fluorine supply in youth may offer women some protection against osteoporosis in menopause. Food sources Most of the fluorine comes from drinking water, in particular in those places where either the fluorine content in the water is naturally high or were it is supplemented to 1 ppm. Seafood contains some fluorine, as do vegetables grown in areas with high soil fluorine. The amount of fluorine introduced into enamel during brushing with F− containing toothpastes is very low, however, some of the toothpaste is invariably swallowed, and this does offer some benefit. Wine and tea are also relatively high in fluorine.

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Uptake and metabolism F− is absorbed with 90 % efficiency, mostly in the stomach. The blood [F− ] is mirrored in other body fluids.

Excretion The blood [F− ] is regulated by the kidneys, about half of the normal daily uptake is excreted.

Deficiency Drinking water [F− ] of less than 1 ppm is associated with a higher incidence of tooth decay. It may also increase the risk of osteoporosis.

Toxicity More than 2.5 ppm of fluorine in drinking water leads to fluorosis with chalky discolourations and brownish stains on enamel. High concentrations of fluorine are cytotoxic (inhibition of enolase → use of fluorine to stop metabolism in blood samples).

Copper Function Copper can occur in the +I and +II oxidation state, and is used in enzymes catalyzing redox-reactions. Cu+ salts tend to be water insoluble and are subject to oxidation under environmental conditions. Hence we can focus here on Cu2+ . The human body contains 70–150 mg of Cu2+ , mostly in bones and muscle. Cu2+ aids the uptake of iron, releases stored iron in the liver and stimulates the synthesis of hemoglobin. Ceruloplasmin (Ferroxidase 1) and Ferroxidase 2 maintain Fe in the ferric (Fe3+ ) state for transport. Cu2+ is found in the active center of monoamine and diamine oxidases, important for the metabolism of serotonin, norepinephrine, dopamine, melanin, tyramine and histamine. Lysyl oxidase is required for the formation of crosslinks in elastin and collagen. Blood clotting factor V also contains Cu2+ . Superoxide dismutase (synonyms are cytocuprein in bone, erythrocuprein in red cells, hepatocuprein in liver or cerebrocuprein in CNS) protects the body against oxidative damage. Cu2+ is also required for the synthesis of phospholipids and in the respiratory chain (cytochrome c oxidase). It is involved in cholesterol metabolism, thermal regulation, immune and cardiac function, although the mechanism is not clear.

Food sources Cu2+ is widely distributed in foods of both animal and plant origin (except milk), some may also leach into the drinking water from copper pipes. Particularly good sources are shellfish, nuts, legumes, whole grain cereals.

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Biochemistry and Genetics

Uptake and metabolism Dietary Cu2+ is absorbed with 25–40 % efficiency in the stomach and upper small intestine by regulated, active transport. In the intestinal cells it is bound to metallothionein. Cu2+ is transported as complex with albumin and transcuprin with the portal blood to the liver, where it is either excreted into bile, stored as metallothionein complex or used for ceruloplasmin synthesis. Ceruloplasmin binds to the cells in the body, where the Cu2+ is absorbed. Uptake is counteracted by excess iron, zinc, molybdenum and ascorbic acid. Excretion in bile, controlled by the adrenal gland. Deficiency Copper deficiency may lead to low ceruloplasmin levels in blood and thereby to normocytic, hypochromic anemia. Leukopenia and neutropenia may also be found. Patients may suffer from osteoporosis and flaring or fractures of the metaphyses and arthritis. Melanin formation will be lower, leading to depigmentation. Arterial, myocardial and neurological disease will be found, in part as consequence from increased cholesterol level. Heart beat may be irregular. Glucose tolerance may be lowered. Toxicity Acute Cu2+ poisoning may be seen after accidental or suicidal intake or from ingestion of acidic foods stored in copper containers. Contact of copper salts with burned skin has also resulted in poisoning. Symptoms are gastric pain, nausea, vomiting, diarrhea, coma, oliguria, hepatic necrosis, vascular collapse and death. Genetic diseases relating to Cu2+ Wilson’s disease (hepatolenticular degeneration) is a chronic Cu2+ poisoning caused by an autosomal, recessive disease (1:20 000 births) in the ATP7B -gene which leads to reduced biliary excretion. The copper accumulates in liver, brain and cornea. This is treated by chelation therapy with penicillamine (usually in combination with Zn2+ and pyridoxin supplements). Provided therapy starts before major tissue damage has occurred, patients may enjoy normal life expectancy. Menke’s kinky hair syndrome, a X-linked genetic disease (1 out of several 100 000 births) results in a defective Cu2+ -ATPase (ATP7A) and is usually fatal by age 3. ATP7Ap resides in the membrane of the Golgi-apparatus and transports Cu2+ from the cytosol into the organelle where it is used to make Cu2+ -dependent enzymes. The enzyme can also move to the plasma membrane to transport excess Cu2+ from the cytosol into the blood. As a consequence of the deficiency copper is not available for its normal functions and accumulates in intestine, spleen, muscle and kidney. The symptoms therefore are a combination of those caused by Cu2+ toxicity and Cu2+ deficiency. Cutis laxa results from a failure to produce lysyl oxidase and therefore functional collagen and elastin.

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Trace elements

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Figure 18.25.: A Kayser-Fleischer-ring is diagnostic for Wilson’s disease. It is caused by copper deposition in Descemet’s membrane. Image from Fred & van Dijk, “Images of Memorable Cases: Case 9,” http://cnx.org/content/m15007/1.2/.

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In Downs syndrome an overproduction of superoxide dismutase may be found. Albinism results from a failure to produce tyrosinase, a Cu2+ dependent enzyme required for melanin biosynthesis. A cytochrome c oxidase deficiency leads to myopathy.

Zinc Function Zinc always occurs in the +II oxidation state. The human body contains about 1.5–2.5 g of Zn2+ , mostly in cytoplasm. There are more than 200 enzymes known which have Zn2+ in the active center, from all 6 main enzyme classes. Zn2+ is required for the incorporation of methionine into skin proteins, important for wound healing. Insulin is stored as Zn2+ complex. Zn2+ is also required for bone development, RNA and DNA polymerases, carboanhydrase, metallopeptidases, NAD+ /NADP+ dependent dehydrogenases (including ethanol metabolism), vitamin A mobilization from liver. Also found in eye, prostrate and semen. Sufficient supply with Zn2+ increases Cd and Pb tolerance. The conversion of dietary folate into the monoglutamate requires Zn2+ . Zn2+ -finger proteins regulate gene expression.

Food sources Highest concentrations are found in seafood, eggs and meat, which may not be available to low income groups. Cereals and legumes are also high in Zn2+ , but cereals contain also phytate, which makes Zn2+ unavailable (reduced by leavening of the bread).

Uptake and metabolism Zn2+ is absorbed in the upper part of the small intestine with 30–50 % efficiency (up-regulated in pregnant females). Absorption is enhanced by the amino acids His and Cys, which form stable, absorbable complexes with Zn2+ . The uptake into the intestinal epithelium is carrier mediated and not energy dependent. In the intestinal cells Zn2+ is bound to metallothionein, in portal blood to albumin. In systemic blood Zn2+ is bound to α-macroglobulin, transferrin and albumin. Albumin-bound Zn seems to be the major transport form to the cells. There is no specific Zn2+ store, and Zn2+ deficiency may develop rapidly. However, short term needs can be bridged by muscle and bone wasting. Chelators like penicillamine, DTPA (used to prevent Fe poisoning in thalassemia), ethambutol (a tuberculostatic) and anticonvulsants like valproate interfere with Zn2+ uptake. Zn2+ uptake efficiency is also reduced in intestinal problems like Crohns disease, short bowel syndrome, jejunoileal bypass, sprue, AIDS-associated diarrhea and in alcoholic cirrhosis.

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Trace elements

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Excretion mainly with pancreas juice, bile and duodenal secretions into feces, but also in urine (increased by EDTA and similar compounds), sweat and (in |) semen or (in ~) menstrual secretions. Hyperzincuria may be found in diabetics (both type 1 and 2). Deficiency About a fourth of the worlds population is estimated to suffer from zinc deficiency. If supply of Zn is below requirements, total Zn concentration in serum (measured for example by atomic absorption spectroscopy AAS) drops only slowly, since most of the Zn is bound tightly in metalloproteins like α-2-macroglobulin. However, the so called labile Zn (free Zn2+ and Zn bound in weak complexes to thiol groups, measurable by fluorescent Zn-chelators like zinquin) drops within a few days. Because of the large number of biochemical processes that require Zn, symptoms of Zn deficiency are variable. Acne, skin lesions, retarded growth, delayed sexual development, oligospermia, delayed wound healing, loss of appetite, loss of taste and smell (hypogeusia and hyposmia). The eye may be affected: photophobia, night blindness, corneal edema and clouding, conjunctivitis, xerosis and keratomalacia, with permanent damage if treatment is late. Hair may be hypopigmented and reddish, with patchy loss. Failure of chemotaxis in monocytes and neutrophiles causes loss of immune function. Patients suffering from Zn2+ deficiency may be irritable, lethargic, depressed, sleepy, with fine tremor, ataxic gait and slurred speech. Conditions like diabetes, asthma, arthritis and sickle cell anemia are worsened by Zn deficiency. Children with Zn2+ deficiency grow slower than their peers, and suffer more frequently from infection. Studies in Vietnam, Bangladesh and Indonesia have shown that Zn2+ supplementation can reduce the incidence of pneumonia by 41 % and diarrhea by 18 %. Both diseases are major causes of infant death. In pregnant females Zn2+ deficiency increases the risk of pregnancy related problems like hypertension, there is also a risk of congenital malformations in the embryo, from low birth weight to neural tube defects like spina bifida and anencephaly. Regionally, Zn2+ deficiency is prevalent in Egypt and Iran, because the diet there contains large amounts of unleavened bread, which contains phytate and interferes with uptake of Zn2+ and other trace metals. Infections with hookworms and Giardia may cause Zn2+ (and Fe) deficiency. Penicillamine interferes with the uptake of Zn2+ , as do high concentrations of Ca, Fe, and Cu. Geophagy (eating of soil) may lead to Zn2+ deficiency, if clay minerals bind this metal.

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Biochemistry and Genetics

Toxicity No short term toxic effects have been seen with uptakes as high as 200 mg/d, but the long term effect of such megadoses is unknown. Very high doses of Zn2+ from galvanized cooking utensils, excess supplementation or industrial pollution leads to fever, vomiting, diarrhea, impaired immune function, Fe loss from liver stores (and consequential microcytic, hypochromic anemia), Cu2+ uptake deficiency and to lowered HDL levels, with a risk of atherogenesis.

Genetic diseases relating to Zn2+ metabolism A rare autosomally determined failure to produce Zn2+ -binding protein in the intestine leads to acrodermatitis enteropathica . The condition can be treated with high oral doses (30–45 mg/d) of Zn2+ , which compensate for the reduced absorption efficiency. Mothers may be unable to secrete Zn2+ into the milk, in these cases infants require Zn2+ supplementation until weaned. Supplementation of the mothers does not help in such cases.

18.3.3. Ultratrace elements Iodine Function The human body contains 15–23 mg of I, 3/4 of this in the thyroid gland. The remainder is found in salivary, mammary and gastric glands and in the kidneys. In severe iodine deficiency, total body I may be less than 1 mg. I is a component of the hormones thyroxin and thyronin. These increase basal metabolism and heat production by up to 30 %, conversion of carotene to vitamin A, protein synthesis, carbohydrate absorption in the intestine, and reproduction. They decrease cholesterol biosynthesis. Because of the stimulation of basal metabolism thyroxin is sometimes used as weight loss aid. This is very dangerous and should not be done!

Food sources Seafood, saltwater fish, seaweed, vegetables grown in I-rich soil. In many countries iodinated table salt is available. In some underdeveloped countries (where salt iodination can not be performed reliably) programs have started where children are given an intramuscular injection of iodine containing poppy seed oil every 2–4 a.

338

18.3.3

Ultratrace elements

Thyroxine Metabolism NH2 OH

NH2 OH H2C C H HC

CH

HC

CH

O

I-

H2C C H

H+ + 2e-

HC

NH2 OH

O

I-

CH

H2C C H

H+ + 2e-

HC

CH

I

I

I

OH

OH

OH 3,5-Diiodotyrosine

3-Monoiodotyrosine

Tyrosine

3,5-DIT

3,5-DIT

Ala

Ala

NH2 OH

NH2 OH H2C C H HC

H2C C H

O

CH

I

I-

I

HC

H+ + 2e-

HC

O

CH

I

I O

O

I

O

CH

HC

CH CH OH

3,5,3'-Triiodothyronine (T3)

CH

I

I OH

Tyroxin (3,5,3',5'-Tetraiodothyronine, T4)

Figure 18.26.: Generation of the hormones T3 and T4 from the amino acid tyrosine in the thyroid gland.

339

18.3.3

Biochemistry and Genetics

Maintaining thyroid hormone concentration in blood: Tyroxin releasing hormone (TRH) Hypothalamus

Thyroid stimulating hormone (TSH) +

Pituitary

Thyroxin +

Thyroid

+

Physiological activity

−

Natural iodine cycle:

Sea water

evaporation (400 kt/a)

precipitation rain

soil

plants

humans

rain and flood extraction

Figure 18.27.: a) The level of thyroxin in blood is tightly regulated. For details see text. b) Global iodine cycle. Iodine evaporated from the seas and rains down onto soil, where it can be taken up by plants. However, extraction of iodine from the soil by rain and flood water is faster than its deposition. For this reason geologically old soil is deficient in iodine, and people living exclusively from plants grown on such soil suffer from iodine deficiency.

340

Ultratrace elements

18.3.3

Uptake and metabolism I is absorbed in the small intestine, and transported in the blood stream to the thyroid (1/3) and the kidneys. Iodine uptake into the thyroid gland is a very efficient secondary active process powered by the Na+ /K+ -ATPase. There is a 100:1 I concentration gradient between thyroidal cells and serum. Release of Thyroxin releasing factor from the hypothalamus increases production of thyroid stimulating hormone in the pituitary (see fig. 18.27a). This in turn stimulates thyroxin release from the thyroid, which in turn reduces the release of thyroid stimulating hormone. Excretion Excess I is transported to the kidneys and the salivary gland. Deficiency There is a natural iodine cycle operating in nature (see fig. 18.27b). Some 400 000 t/a of iodine evaporate from the seas and are transported to the soil with rain. However, this is not sufficient to replace the loss from the soil by rain and flood extractions. Geologically old soil, or soil in flood plains, tends to be poor in iodine, and the plants growing on those soils will have a low I content too. People living in those areas, and obtaining their food locally may become I deficient. Iodine deficiency causes (simple) goiter, a hyperplasia of the thyroid gland, which can grow up to 1 kg in weight instead of the normal 15–25g, leading to breathing difficulties. This may be because of insufficient I supply or the presence of goitrogenic substances like SCN− , which interfere with I metabolism. Such substances are found in raw cabbage, peaches, almonds, soybeans, cassava and peanuts. Some pharmaceuticals also are goitrogenic like sulphonamides and para-aminobenzoic acid. Eventually iodine deficiency will lead to mental retardation, in extreme cases to cretinism. There are about 1 × 109 people worldwide suffering from iodine deficiency, 200 × 106 people suffering from simple goiter (of which 4 % are caused by goitrogenic chemicals). 25 × 106 people worldwide suffer from mental defects resulting from gross iodine deficiency, of those 5 × 106 are suffering from overt cretinism. This makes I deficiency a significant health problem especially in developing countries, which is shameful as it can be prevented easily and with small financial input by adding traces of sodium iodate to cooking salt. Low I supply during pregnancy leads to cretinism in the infant (dwarfed, mentally retarded, thick and dry skin, protruding abdomen). This can be treated only soon after birth, otherwise permanent damage occurs. Stillbirths are also common. If I is low during childhood, myxedema may develop in adults, characterized by sparse hair, dry and yellow skin, low voice, psychomotor defects and poor cold tolerance. Newer research indicates that this may actually be a condition caused by a combined iodine and selenium deficiency. The enzymes that produce and inactivate T3 and T4 contain Se in their active center.

341

18.3.3

Biochemistry and Genetics

Figure 18.28.: Left: Goiter is the result of iodine deficiency and mainly found in isolated populations with little trade with the outside world. Middle: Myxedema can be caused by hypo- and hyperthyroidism, the mechanism is unclear. Right: The swellings are caused by incorporation of excess mucopolysaccharides, which stain heavily with alcian blue. Pictures from the archives of Martin Finborud (1861-1930), J. Chung-Leddon, Dermatol. Online J. 7:1 (2001) and Hunzeker et al., Dermatol. Online J. 14:10 (2008)

Toxicity In some populations even very high dietary iodine intakes seem to be well tolerated (up to 80 mg/d in Japanese communities where seaweed is a major part of the diet). In other populations uptake as low as 0.1 mg/d may cause problems. Acute increase of I supply leads to a repression of thyroid hormone synthesis ( WolffChaikoff-effect ). If the high supply persists, iodine transport into the thyroid is reduced to allow normal hormone production rates. In some cases this adaptation does not occur, leading to goiter and iodine myxedema, sometimes also to inflammation of the salivary glands (sialoadenitis) and mouth sores. Molybdenum and tungsten Function 9 mg of Mo are found in the human body, in liver, kidneys, the adrenals and in red blood cells. Mo is a cofactor in the metabolism of C, N and S, it occurs for example in xanthine oxidase, sulphite oxidase and aldehyde oxidase. It promotes the retention of F− and therefore prevents tooth decay. Mo is a very rare element in the earths crust, but because of its good water solubility it is the most abundant transition metal in sea water, from which early life emerged. It can occur in the oxidation states IV, V and VI, and thus catalyze the transfer of single

342

18.3.3

Ultratrace elements + [O] Cys O

S

N

HN H2N

H

N

N

S

O Mo OH S

O

H MoCo

C H2

O Mo

IV

Mo

VI

O O P O R O H2O

2 [H]

Figure 18.29.: Molybdenum cofactor (MoCo) bound to an enzyme. This cofactor occurs in oxidoreductases like xanthin, sulphite and aldehyde oxidase, nitrate and DMSO reductase. Molybdenum can be oxidized by binding an oxygen atom, and it can be reduced by giving up that oxygen to hydrogen. This reduction can happen in one step or in two, as Mo can also have the oxidation number V. electrons and of electron pairs. Because of that property, it can transmit between single electron and electron pair transfer reactions. These properties make Mo an ideal catalyst for living organisms. W is used in much the same way as Mo, but it forms more stable bonds with sulphur and is more oxygen sensitive than Mo. Thus it is used in thermophilic anaerobic organisms, while Mo is used preferentially in aerobic organisms living in moderate temperatures (like man). Food sources Legumes, meat and to a lesser extend whole grain cereals. Uptake and metabolism Absorbed with 25–80 % efficiency in stomach and small intestine. Transported in blood loosely bound to erythrocytes and α2 -macroglobulin. Stored in the liver as “Mo cofactor” di(carboxaminomethyl)molybdopterine (MoCo, see fig. 18.29). Excretion mainly in urine as molybdate, some also in bile. Mo excretion is promoted by sulfate. Deficiency Low appetite, slow growth, reduced fertility. High mortality of infant and mother. Mental disturbancies, coma, death. Toxicity Diarrhea, anemia, slow growth, failure of red cell maturation, gout. Prevents utilization of Cu2+ .

343

18.3.3

Biochemistry and Genetics

Genetic diseases relating to Mo A (fortunately) rare condition is the inability to synthesize MoCo (autosomal recessive inheritance). Most frequent is a defect in the MOCS1A or MOCS1B genes, which prevents the synthesis of the pterin “precursor Z” from GTP (MoCo deficiency type A). MoCo deficiency leads to the accumulation of toxic metabolites in the body, as a consequence neurological abnormalities appear (increased muscle tone, rigid posture, seizures) leading to death in early infancy. An experimental treatment of type A MoCo deficiency by i.p. or i.v. injection of precursor Z isolated from E. coli has recently been described. No treatment is known for type B and C MoCo deficiency, since MoCo itself is too unstable for isolation. Selenium Function 15mg of Se are stored in the human body, mainly in the liver. Se as Sec is an essential component in several enzymes: glutathione peroxidase (cGPx) is important for protection against oxidants, double-minus mice show increased sensitivity against poisons like Paraquat and Diquat, which generate peroxides. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) Function is not quite clear. Could possibly be involved in the regulation of prostaglandin- and leucotrien synthesis by regulating the peroxide tonus in the cell. PHGPx has an additional function in sperm production: It co-polymerizes with SH-containing proteins to form the capsula (thus becoming a structural protein rather than an enzyme). Selenoprotein P in plasma, with unknown function. It contains 11 Sec groups and could be a scavenger for peroxides. Iodothyronine deiodinases activate Tyr to T3 and deactivate T3 and T4 in the thyroid gland. The iodination reaction requires peroxide, thus thyreocytes express also cGPx and PHGPx to balance the peroxide tonus. Myxedematous cretinism seems to be caused by a combined iodine and selenium deficiency. Thioredoxin reductases DNA-synthesis (co-substrates of ribonucleotide reductase), antioxidant. Knock-out mice die in early embryonal development. Selenophosphate synthase Synthesis of the seleno carrier protein required for the synthesis of Sec tRNA. Other possible functions of Se include liver function and energy metabolism. At least in bacteria, Se may occur as 5-methylaminomethyl-2-selenouridine in some tRNAs. In animals high Se protects against some cancers, but human studies so far were inconclusive. Se may also offer some protection against chemicals like Paraquat, Hg, Cd and Ag.

344

18.3.3

Ultratrace elements

In the vicinity of the Sec in selenoproteins a Trp- and a Gln-residue will be found, these lead to the deprotonation of the selenol group to −Se− . This group is able to reduce for example H2 O2 with very high speed (in excess of 10 × 107 M−1 sec−1 ) and is later recycled by thiols. The reason for the incorporation of Sec into the active center of proteins instead of Cys is the much lower pKa value of the −SeH group (5.2) compared to the −SH group (8.2). The reaction proceeds as follows: +XOOH

+GSH

+GSH

−OH −

−XOH

−GSSG

R−SeH GGGGGGGGA R−Se− GGGGGGGGA R−Se−OX GGGGGGGGA R−Se−S−G GGGGGGGGA R−SeH −H +

At physiological pH (7.4) the selenol-group is fully ionized, the thiol group is not. Consequently experimental replacement of Sec with Cys in peroxidases leads to an enzyme that is several 100-fold less active and has a pH-optimum of about 9.

Food sources Organ meats, seafood, muscle meats, cereals and dairy products (in order of decreasing concentration). Se content is reduced by milling of cereals and cooking. Plant Se content depend on the Se content of the soil in which they were grown.

Uptake and metabolism In plants both Sec and Se-Met are produced unregulated as a consequence of the Se content in soil. Animals do not synthesize Se-Met, after uptake this amino acid is either metabolized or build into proteins randomly instead of Met. Sec however is synthesized in a regulated fashion. For this, the hydroxy-group of SertRNAsec is phosphorylated by a specific kinase, the phosphate is than exchanged against a HSe-group from a reduced Se carrier protein. This tRNA is complementary to the UGA stop codon, incorporation of Sec requires a special translation factor with high homology to EF-Tu. It is not entirely clear when the Opal -codon is interpreted as “stop translation” and when as “Sec”, apparently special stem-loop structures in the mRNA in the 3’-untranslated region control this from a distance. Absorption efficiency of dietary Se ranges from 50 % for inorganic Se compounds to 100 % for Se-Met. Uptake and transport mechanism are unclear. There is essentially no free Sec (not incorporated in proteins) in our body, as it gets rapidly destroyed by Sec β-lyase.

Excretion Se is normally excreted in urine in a variety of compounds including trimethyl selonium. High doses of Se lead to the formation of dimethyl selenide, which is breathed out, giving the breath a characteristic, radish like smell.

345

18.3.3

Biochemistry and Genetics

NH2 O HO C C C H2 H O Rib Ade

Pi

ATP

NH2 O O C C C H2 H O Rib Ade

ADP

ACU

ACU Ser-tRNAsec (complementary to the UGA "opal" stop codon)

Selenium carrier protein Pi

H

Se

NH2 O C C C H2 H O Rib Ade

ACU Se-Cys tRNA

Figure 18.30.: In mammals selenocysteine is synthesized on its tRNA. This tRNA is complementary to the opal stop codon. Unlike selenocysteine selenomethionine is build into proteins randomly by competition with methionine.

346

Objectives in summary

18.4

Deficiency Heart muscle degeneration (Cardiomyopathy, Keshan disease) characterized by multifocal necrosis, fibrous replacement, myocytolysis. Se supplementation can prevent the progress of the disease, but not cure preexisting damage. In the CGPx(-/-) mouse non-pathogenic Coxsackie-virus quickly mutate to virulent strains because of the increased hydroperoxide tonus, these cause heart conditions very much like those seen in Keshan disease. Se-deficiency is also a possible cause of Kashin-Beck disease, an osteoarthritis in preadolescence. The disease is characterized by necrotic degeneration of chondrocytes, dwarfism and joint deformation. General symptoms include muscle pain, defective finger nails and red blood cells. Other (co)-causes may include iodine deficiency and mycotoxins from spoiled food. Toxicity Intakes between 24–200 µg/d do not seem to cause problems. However, chronic intakes in excess of that lead to dermatitis, loss of hair, nail deformation and loss (alkali disease). Acute intoxication leads to nausea, diarrhea, irritability, fatigue, peripheral neuropathy, and vomiting. This seems to be caused by the formation of dimethyl selenide. In animal studies high Se increases the sensitivity to aflatoxin and acetaminophen. Excess Se is also carcinogenic.

18.4. Objectives in summary At the end of this course, students should be able to • explain the dose-effect relationships in micronutrients, using the terms estimated average requirement (EAR), recommended dietary allowance (RDA), tolerated upper intake level (UL), Dietary reference intake (DRI), adequate intake (AI) and US recommended daily allowance (US-RDA). • define the terms enrichment, fortification, supplementation, vitamin, anti-vitamin, provitamin, mass element, trace element and ultratrace element. • explain how the solubility of vitamins in fat or water determines their toxicity and the risk for the development of deficiencies. • explain the function of the various vitamins, their food sources, the signs and symptoms of deficiencies and toxicity and the diseases associated with their metabolism. • explain the function of the various minerals, their food sources, the signs and symptoms of deficiencies and toxicity and the diseases associated with their metabolism. • appreciate the role of a balanced nutrition.

347

19. Carbohydrate Metabolism 19.1. Gluconeogenesis Gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors. It occurs in liver and kidney and is necessary to maintain the blood glucose level when dietary carbohydrates are in short supply and glycogen reserves are depleted. It uses the reversible reactions of glycolysis, but irreversible pyruvate kinase, phosphofructokinase, and glucokinase (hexokinase) reactions have to be bypassed.

19.1.1. The first bypass: From pyruvate to phosphoenolpyruvate (PEP)

ATP HCO3

Pyruvate

ADP

CO2

Pi

GTP

GDP Phosphoenolpyruvate

Oxaloacetate

These two reactions bypass the pyruvate kinase reaction of glycolysis. The input of two high-energy phosphate bonds makes the formation of PEP from pyruvate energetically possible.

19.1.2. The second and third bypasses Phosphofructokinase and glucokinase are bypassed by fructose 1,6-bisphosphatase and glucose 6-phosphatase:

349

19.1.5

Biochemistry and Genetics Glycolysis

Gluconeogenesis

ATP

Pi

Glucose

Glucose-6-phosphatase

Glucokinase

ADP

Glucose-6-phosphate

ATP

Fructose-6-phosphate

Phosphofructokinase

Pi Fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphate

ADP

19.1.3. Substrates of gluconeogenesis 3.

Substrates of gluconeogenesis

Glucose Amino Acids Glycerol Aspartate Lactate

Oxaloacetate Pyruvate

TCA cycle intermediates Alanine

Acetyl-CoA and fatty acids are not substrates of gluconeogenesis! Acetyl-CoA and fatty acids are not substrates of gluconeogenesis! 4. Energy balance 19.1.4. Energy balance

The formation of 1 molecule of glucose from 2 molecules of lactate requires: 2 ATP by pyruvate carboxylase The formation of 1 molecule of glucose from 2 molecules of lactate requires: 2 GTP by PEP carboxykinase 2 ATP pyruvate carboxylase 2 ATP by by phosphoglycerate kinase

2 2 6 5. The

GTP by PEP carboxykinase Theby energy for gluconeogenesis ATP phosphoglycerate kinasecomes from fatty acid oxidation. high energy phosphates Regulation of gluconeogenesis energy for gluconeogenesis comes from fatty acid oxidation.

1. Adaptive control: The levels of glycolytic enzymes are high in the well-fed state (high insulin), levels of gluconeogenic enzymes are high in the fasting state (high glucagon). 19.1.5.2. Regulation of gluconeogenesis Allosteric effectors: ATP, citrate, and acetyl-CoA favor gluconeogenesis, AMP and ADP favor glycolysis. 3. control: Phosphorylation: hormonal effects mediated enzyme Adaptive The levelsShort-term of glycolytic enzymes areare high in the by well-fed state (high phosphorylation. insulin), levels of gluconeogenic enzymes are high in the fasting state (high glucagon). - Pyruvate kinase is inactivated by cAMP-induced phosphorylation. - Insulin increases and glucagon decreases the concentration of fructose 2, 6-bisphosphate, an allosteric activator of phosphofructokinase and inhibitor of fructose 1,6-bisphosphatase.

350

II.

GLYCOGEN METABOLISM

Glycogen is stored in most cells of the body but is most plentiful in liver (up to 8%) and muscle (1%). Liver glycogen is a carbohydrate reserved for the maintenance of blood glucose; muscle glycogen is used only by the muscle itself during exercise. 1. Structure A polysaccharide of glucose residues linked by α-1, 4 glycosidic bonds. Highly

19.2.3

Synthesis

Allosteric effectors: ATP, citrate, and acetyl-CoA favor gluconeogenesis, AMP and ADP favor glycolysis. Phosphorylation: Short-term hormonal effects are mediated by enzyme phosphorylation. Pyruvate kinase is inactivated by cAMP-induced phosphorylation. Insulin increases and glucagon decreases the concentration of fructose-2,6-bisphosphate, an allosteric activator of phosphofructokinase and inhibitor of fructose 1,6-bisphosphatase.

19.2. Glycogen Metabolism Glycogen is stored in most cells of the body but is most plentiful in liver (up to 8 %) and muscle (1 %). Liver glycogen is a carbohydrate reserved for the maintenance of blood glucose; muscle glycogen is used only by the muscle itself during exercise.

19.2.1. Structure A polysaccharide of glucose residues linked by α-1,4-glycosidic bonds. Highly branched (one branch point every 12 residues) with α-1,6-glycosidic bonds at the branch points.

19.2.2. Synthesis 2. Synthesis Phosphoglucomutase Glucose 6-phosphate

Glucose 1-phosphate UTP Glucose 1-phosphate uridyl-transferase UDP

(Glc)n+1

(Glc)n

PPi UDP-glucose

Glycogen Synthase

At equilibrium about 5% of all glucose phosphate is glucose 1-phosphate and

At equilibrium about 5 % of glucose phosphate is glucose 1-phosphate and 95 % is glu95% is glucose 6-phosphate. Theall bond between UDP and glucose is moderately energy-rich (3-4 kcal/mol). cose 6-phosphate. The bond between UDP and glucose is moderately energy-rich (12– Glycogen synthase makes the α-1, 4 glycosidic bonds. The branches with their 17 kJ/mol). α-1, 6-glycosidic bonds are made by a branching enzyme which transfers an

oligosaccharide from the nonreducing end of the moleculebonds. to a C-6The carbon. Glycogen synthase makes the α-1,4-glycosidic branches with their α-1,6-glycosidic bonds are made by a branching enzyme which transfers an oligosaccharide from the non3. Degradation. Glycogen removes reducing end of phosphorylase the molecule to a C-6 glucose carbon.residues by phosphorolytic cleavage from the nonreducing end, forming glucose 1-phosphate. It cleaves only α-1, 4-glycosidic bonds. The branches are removed by a debranching enzyme which transfers pieces of 3 glucose units from the branch points and hydrolyzes the α-1, 6glycosidic bonds. Because of hydrolysis of the α-1, 6 bonds 8% of the glucose in glycogen is released as free glucose rather than glucose 1-phosphate. 4.

Difference between liver and muscle The liver has glucose 6-phosphatase. Therefore it makes free glucose from glycogen via glucose 1-phosphate and glucose 6-phosphate. The liver synthesizes glycogen after a carbohydrate-rich meal and degrades it between meals to maintain the blood glucose level. Liver glycogen lasts for 12 – 24 h. Muscle has no glucose 6-phosphatase. Therefore muscle glycogen can be used only locally, for glycolysis. It lasts for 2 hours during a marathon race.

351

19.2.5

Biochemistry and Genetics

19.2.3. Degradation Glycogen phosphorylase removes glucose residues by phosphorolytic cleavage from the nonreducing end, forming glucose-1-phosphate. It cleaves only α-1,4-glycosidic bonds. The branches are removed by a debranching enzyme which transfers pieces of 3 glucose units from the branch points and hydrolyzes the α-1,6-glycosidic bonds. Because of hydrolysis of the α-1,6 bonds 8 % of the glucose in glycogen is released as free glucose rather than glucose-1-phosphate.

19.2.4. Difference between liver and muscle The liver has glucose-6-phosphatase. Therefore it makes free glucose from glycogen via glucose-1-phosphate and glucose-6-phosphate. The liver synthesizes glycogen after a carbohydraterich meal and degrades it between meals to maintain the blood glucose level. Liver glycogen lasts for 12–24 h. Muscle has no glucose-6-phosphatase. Therefore muscle glycogen can be used only locally, for glycolysis. It lasts for 2 h during a marathon race.

19.2.5. Regulation of glycogen metabolism The enzymes of glycogen metabolism are controlled by: • Hormone-induced phosphorylation. • Allosteric effectors.

Glycogen synthase is inactivated and glycogen phosphorylase is activated by phosphorylation:

Glycogen synthase is inactivated and glycogen phosphorylase is activated by phosphorylation: ATP

-

ADP

Glucose 6-phosphate

Protein Kinase Glycogen synthase a (dephosph'd: active)

Glycogen synthase b (phosph'd: less active) Protein Phosphatase

++

Ca

+

Pi

AMP (in muscle)

-

-

Glucose 6-phosphate ATP ADP Glucose (in muscle) (in liver) Protein Kinase Glycogen phosphorylase b Glycogen phosphorylase a (dephosph'd: less active) (phosph'd: active) Protein Phosphatase Pi

352

Glucose (liver), AMP (extrahepatic tissues), and glucose 6-phosphate are allosteric effectors - indicated effects are shown with gray arrows above. Phosphorylation is induced by calcium and cAMP. In the liver, cAMP is elevated by glucagon and calcium by epinephrine (α-receptors). In muscle, cAMP is elevated by epinephrine (β-receptors), and calcium is elevated during excitationcontraction coupling. Dephosphorylation is effected by phosphatase-1 which is stimulated by insulin and inhibited by cAMP. Insulin also antagonizes glucagon directly by lowering the cAMP concentration. INSULIN Plasma

Glucagon (liver) Epinephrine (muscle, liver)

Acetylcholine (muscle) Epinephrine (liver)

+ Glycogen storage diseases

AMP (in muscle)

-

Glucose 6-phosphate ATP (in muscle) Protein Kinase Glycogen phosphorylase b (dephosph'd: less active) 19.2.6 Protein Phosphatase Pi

Glucose (liver), AMP (extrahepatic tissues), and glucose-6-phosphate are allosteric effectors Glucose (liver), AMP (extrahepatic tissu allosteric effectors - indicated effects are shown wi - indicated effects are shown with gray arrows above. Phosphorylation is induced by calcium

by glucagon and calcium by epinephrin Phosphorylation is induced by calcium and cAMP. In the liver, cAMP is elevated elevated by elevated by epinephrine (β-receptors), and ca glucagon and calcium by epinephrine (β-receptors). In muscle, cAMP is elevatedcontraction by epinephrine coupling. Dephosphorylation is e stimulated by insulin and inhibited by cAMP. Insu (β-receptors), and calcium is elevated during excitation-contraction coupling. Dephosphoryby lowering the cAMP concentration. lation is effected by phosphatase-1 which is stimulated by insulin and inhibited by cAMP. InINSULIN

Glucagon (liver) Epinephrine (muscle, liv

Plasma Membrane

+

+ cAMP

Phosphatase-1

+ Protein Kinase A

+

+

Phosphorylase Kina

-

+

Glycogen Phosphorylase

sulin also antagonizes glucagon directly by lowering the cAMP concentration.

+ 156

19.2.6. Glycogen storage diseases Problem: Recessively inherited deficiency of a glycogen degrading enzyme, with accumulation of glycogen. 3 types: Hepatic: Hepatomegaly, fasting hypoglycemia Myopathic: Muscle weakness, muscle cramps on exertion. Generalized: Brain or myocardium are also affected, besides liver and skeletal muscle. Von Gierke’s disease (type I): Deficiency of glucose-6-phosphatase in liver and kidney. Severe hepatomegaly and fasting hypoglycemia, ketosis, hyperuricemia, lactic acidosis. Treated with regular carbohydrate feeding. McArdle’s disease (type V): Deficiency of glycogen phosphorylase in muscle. Muscle pain and cramps on exertion, sometimes intermittent myoglobinuria, muscle wasting in some older patients. Little or no increase of blood lactate after exercise. No treatment required.

353

Glyc

6. Glycogen storage diseases inherited deficiency of a glycogen degrading enzyme, Biochemistry and Genetics 3 types: Hepatic: Hepatomegaly, fasting hypoglycemia Myopathic: Muscle weakness, muscle cramps on exertion. Generalized: Brain myocardium are affected, besides liver and Pompe’s disease (typeskeletal II): or muscle. Deficiency ofalso a lysosomal α-1,4-glucosidase Problem: Recessively 19.3.1 with accumulation of glycogen.

(‘acid maltase’) in all tissues. Glycogen accumulates in lysosomes. Cardiac involvement, with death before a) Von Gierke’s disease (type I): Deficiency of glucose 6-phosphatase in liver ageand2 a. kidney. Severe hepatomegaly and fasting hypoglycemia, ketosis, hyperuricemia, lactic acidosis. Treated with regular carbohydrate feeding. b) McArdle’s disease (type V): Deficiency of glycogen phosphorylase in muscle. Muscle pain and cramps on exertion, sometimes intermittent myoglobinuria, muscle wasting in some older patients. Little or no increase of blood lactate after exercise. No treatment required.

19.3. Dietary Fructose and Galactose

c) Pompe’s disease (type II): Deficiency of a lysosomal α-1, 4-glucosidase (‘acid maltase’) in all tissues. Glycogen accumulates in lysosomes. Cardiac The liver is with the death mostbefore important site of fructose and galactose metabolism. involvement, age 2. III.

DIETARY FRUCTOSE AND GALATOSE

liver is the most important site of fructose and galactose metabolism. 19.3.1.TheFructose metabolism 1.

Fructose metabolism Only a small fraction of the dietary fructose is phosphorylated by hexokinase in Only a smalltissues. fractionInofliver, thekidney dietary phosphorylated by hexokinase in extrahepatic extrahepatic andfructose intestine, is fructose is phosphorylated by fructokinase: tissues. In liver, kidney and intestine, fructose is phosphorylated by fructokinase: Glucose

Fructose Pi

ATP Fructokinase ADP

Glucose 6-Phosphate

Fructose 1-Phosphate Aldolase B

Fructose 6-Phosphate Pi

Dihydroxyacetone- Phosphate

Glyceraldehyde

Aldolase

ATP ADP Glyceraldehyde 3-Phosphate

Fructose 1,6-bis-Phosphate Lactate

Pyruvate

Triglyceride

Acetyl-CoA

157

Fructose is glycolyzed more rapidly than glucose because phosphofructokinase is bypassed. Also, fructose-1-phosphate can accumulate in the liver because fructokinase has a higher activity than aldolase B. The use of fructose instead of glucose in parenteral nutrition causes lactic acidosis, liver damage and hypertriglyceridemia. Essential fructosuria is a benign condition, caused by an inherited deficiency of fructokinase. Fructose is high in blood and urine after a fructose-containing meal. Aldolase B deficiency leads to hereditary fructose intolerance (HFI), with hypoglycemia and nausea after eating fructose. Liver damage is possible, but affected children develop a strong aversion to sweets, and affected adults have very good teeth.

354

Fructose is glycolyzed more rapidly than glucose because phosphofructokinase is bypassed. Also, fructose 1-phosphate can accumulate in the liver because fructokinase has a higher activity than aldolase B. The use of fructose instead of glucose in parenteral nutrition causes lactic acidosis, liver damage and hypertriglyceridemia. Reactions 19.4.1 Essential fructosuria is a benign condition, caused by an inherited deficiency of fructokinase. Fructose is high in blood and urine after a fructose-containing meal. Aldolase B deficiency leads to hereditary fructose intolerance (HFI), with hypoglycemia and nausea after eating fructose. Liver damage is possible, but affected Fructose-1,6-bisphosphatase deficiency also leads children develop a strong aversion to sweets, and affected adults have to veryfructose good teeth.intolerance, but patients Fructose 1, 6-bisphosphatase leads to intolerance fructose intolerance, also have hypoglycemia. In the deficiency 2 forms ofalso fructose phosphorylated sugars build but patients also have hypoglycemia. In the 2 forms of fructose intolerance up in the liver.sugars Thisbuild depletes inorganic causing livercausing damage. Fructose-1-phosphate phosphorylated up in the liver. This phosphate, depletes inorganic phosphate, liver damage.glucokinase Fructose 1-phosphate stimulates glucokinase and inhibits glycogen stimulates and inhibits glycogen phosphorylase, causing hypoglycemia. phosphorylase, causing hypoglycemia. 2. Galactose metabolism Pathway: Galactose metabolism 19.3.2.

Galactose ATP Galactokinase ADP Galactose 1-Phosphate UDP-glucose Galactose 1-Phosphate Uridyl-transferase Glucose 1-Phosphate UDP-galactose UDP-galactose-4-epimerase Glucose 6-Phosphate

UDP-glucose

the absence of dietarygalactose, galactose, the epimerase reaction supplies supplies UDP-galactose In the Inabsence of dietary thereversible reversible epimerase reaction UDP-galactose for biosynthetic reactions. for biosynthetic Galactosemiareactions. is caused by an inherited deficiency of galactose 1-phosphateuridyl-transferase. Vomiting, jaundice and CNS dysfunction develop within weeks after birth. Later: Liver failure, cataracts (lens opacities), mental deficiency. The Galactosemia caused1-phosphate by an inherited galactose-1-phosphate-uridyl-transferase. accumulation of is galactose causes deficiency liver damage of (depletion of inorganic

Vomiting, jaundice and CNS dysfunction develop within weeks after birth. Later: Liver failure, cataracts (lens opacities), mental deficiency. The accumulation of galactose-1-phosphate 158 causes liver damage (depletion of inorganic phosphate!); galactitol, formed by aldose reductase in the lens, causes cataracts. Patients have reducing sugar in the urine, but enzymatic tests for glucose are negative. A milk-free diet permits normal development.

19.4. The Pentose Phosphate Pathway 19.4.1. Reactions The pentose phosphate pathway oxidizes glucose with formation of NADPH + H+ , used for reductive biosynthesis and the antioxidant defenses of the cell. It also produces ribose5-phosphate, a precursor for nucleotide synthesis. It has an oxidative and a nonoxidative

355

19.4.3

Biochemistry and Genetics

branch. Reactions of the oxidative branch: +

H2 O3P O C

NADP

+

H NADPH

H2 O3P O C

O

O

OH

O

OH OH

OH

Glc-6-P dehydrogenase

OH

OH

OH 6-P-gluconolactone

Glc-6-P

H2O

CO2

H

+

+

H2C OH

H NADPH

NADP

+

O C HC OH HC OH

COO

-

HC OH HO CH

6-P-gluconate dehydrogenase

C O PO3 H2 Ribulose-5-P

HC OH HC OH C O PO3 H2 6-P-gluconate

The nonoxidative branch links ribulose-5-phosphate to glycolysis and gluconeogenesis in a sequence of freely reversible reactions. Transaldolase and the thiamine-dependent transketolase are the most important enzymes.

19.4.2. Products and regulation For each CO2 released, the oxidative branch forms 2 NADPH + H+ . The reactions of the oxidative branch are irreversible, therefore they can maintain a high cellular [NADPH + H+ ]/ [NADP+ ] ratio. Glucose-6-phosphate dehydrogenase is inhibited by a high [NADPH + H+ ]/ [NADP+ ] ratio. There is also enzyme induction in the well-fed state.

19.4.3. Physiological role High pentose phosphate pathway activity is seen in tissues that make reductive biosynthesis (liver, adipose tissue, lactating mammary gland, adrenal cortex) and in tissues exposed to oxidative stress (cornea, RBCs).

356

Glucose-6-phosphate dehydrogenase deficiency

19.5.1

When the cell needs a lot of NADPH + H+ , the products of the nonoxidative branch are recycled to glucose-6-phosphate, and the cycle can repeat itself. When the cell needs a lot of ribose, ribose-5-phosphate is formed not only by the oxidative branch, but also through the reversible reactions of the nonoxidative branch.

19.4.4. Glucose-6-phosphate dehydrogenase deficiency

A partial deficiency of glucose-6-phosphate dehydrogenase in red blood cells is common in Africa, the middle East, and South Asia. X-linked recessive inheritance. Asymptomatic, but acute hemolysis develops in response to primaquine (an antimalarial) and some other drugs; also after eating broad beans (favism). About 1 × 108 males are affected worldwide, including 11 % of black Americans.

RBCs require NADPH + H+ for protection from oxidative damage. NADPH + H+ maintains the tripeptide glutathione in the reduced state. Glutathione can be used to destroy hydrogen peroxide (H2 O2 ) in the glutathione peroxidase reaction, reduced glutathione has to be regenerated by the NADPH + H+ -dependent enzyme glutathione reductase: H2O2

2 H2O

Glutathion peroxidase

γGlu Cys Gly

SH

+

γGlu

γGlu

HS Cys

Cys

Gly

Gly

γGlu S

S Cys Gly

Glutathion reductase

NADP

+

NADPH +

H

357

19.6

Biochemistry and Genetics

19.5. The ‘Minor’ Pathways 19.5.1. The Polyol Pathway The polyol pathway provides an endogenous source of fructose. Fructose occurs, for example, in seminal fluid in concentrations up to 200 mg/dL. HC

O

HC OH

+

H NADPH

NADP

HC OH

+

HC OH

+

NAD

H NADH

HO CH

HO CH HC OH

+

H2C OH

Aldose reductase

HC OH HC OH

D-glucose

C O HO CH

Sorbitol dehydrogenase

C OH H2

C OH H2

H2C OH

D-sorbitol

HC OH HC OH C OH H2 D-fructose

19.5.2. Synthesis of Amino Sugars Amino sugars are components of glycolipids, glycoproteins and proteoglycans. They can be synthesized in the body: The nitrogen is derived from the side-chain of glutamine. The acetyl group on the nitrogen of many amino sugars comes from acetyl-CoA. UDP-derivatives are the activated forms of the amino sugars for biosynthetic reactions.

19.5.3. The uronic acid pathway. Glucuronic acid is synthesized from glucose in an NAD+ -dependent reaction. It is a constituent of glycosaminoglycans, and in the liver it is used for the conjugation of bilirubin and some drugs. The enzymes of the glucuronic acid pathway are induced by many drugs in the liver.

19.6. Practice Questions The liver has to maintain an adequate blood glucose level in the fasting state. Try to predict the effects of inherited deficiencies of liver enzymes on the fasting blood glucose level: 1. Glucokinase

358

Objectives in Summary

19.7

2. Glycogen phosphorylase 3. Glucose 6-phosphatase 4. Fructose 1, 6-bisphosphatase 5. Aldolase B After eating 100 g of fructose, the blood levels of lactic acid and triglycerides are higher than after 100 g of glucose. Why? Assume that pyruvate dehydrogenase is deficient in all cells. Which tissue would suffer most? Arsenite, the most toxic form of arsenic, binds tightly to the two sulfhydryl groups in dihydrolipoic acid and thereby inhibits lipoic acid dependent reactions. Which metabolites are elevated in the blood after arsenite poisoning? Can arsenic be determined in dead bodies? Which metabolites accumulate in the blood of patients with thiamine deficiency, and how would you use this for the diagnosis of thiamine deficiency? Which RBC enzyme can be assayed to test for thiamine deficiency, and how would you design such a test? How does cyanide poisoning affect cellular energy charge, [NADH + H+ ]/ [NAD+ ] ratio, the oxidation state of respiratory chain components, TCA cycle activity, glycogen metabolism and glycolysis, blood pH and body temperature? Would poisoning with pentachlorophenol (a wood preservative that uncouples oxidative phosphorylation) do the same?

19.7. Objectives in Summary 1. Name the organs of gluconeogenesis and the physiological conditions in which gluconeogenesis is important. 2. Identify the reactions of gluconeogenesis that bypass the irreversible reactions of glycolysis. 3. State the energy requirements of gluconeogenesis, and name important metabolic processes that supply this energy in the gluconeogenic tissues. 4. Describe the effects of hormones and metabolites on gluconeogenesis, both in short term and long term and predict the clinical effects of deficiencies of individual gluconeogenic enzymes. 5. Describe the functions of glycogen in liver, muscle and other tissues.

359

19.7

Biochemistry and Genetics

6. List the sequence of intermediates in glycogen synthesis and glycogen degradation, with special emphasis on effects of metabolites and of phosphorylation/dephosphorylation on the catalytic activities of glycogen synthase and glycogen phosphorylase. 7. Predict the clinical effects of deficiencies of glycogen degrading enzymes in liver and muscle. 8. List the sequence of reactions by which fructose and galactose are channeled into the glycolytic pathway, and the major tissues where these reactions take place. 9. Describe the clinical presentations and the treatment for patients with deficiencies for fructose and galactose metabolizing enzymes. 10. Name the most important product of the oxidative branch of the pentose phosphate pathway, its role in metabolism and the consequences of a partial deficiency of glucose6-phosphate dehydrogenase in red blood cells. 11. Know that subjects with an atypical form of transketolase and thiamine deficiency get Wernicke-Korsakoff syndrome, and that amino sugars and glucuronic acid can be synthesized endogenously from phosphorylated monosaccharides.

360

20. Lipid Metabolism

20.1. Structures

Most naturally occurring fatty acids are unbranched and have an even number of carbons. The carbons are either numbered, or they are designated by Greek letters. The last carbon in the chain is called the ω (omega) carbon: H3C

(CH2)16 COOH

=

β

ω

H3C Stearic acid

COOH α

The carbons of saturated fatty acids are linked only by single bonds. Mono-unsaturated fatty acids have one C=C double bond, and polyunsaturated fatty acids have more than one. Positions of double bonds are specified by their distance from the carboxy end. A ∆9 double bond, for example, is between carbons 9 and 10.

Humans cannot introduce double bonds beyond position ∆9. Therefore some polyunsaturated fatty acids, notably linoleic acid and linolenic acid are nutritionally essential. The biosynthetic class of these fatty acids is defined by the position of the double bond closest to the omega carbon.

361

20.2

Biochemistry and Genetics

Examples of saturated fatty acids 4

Butyric acid

CH3-(CH2)2-COOH

14

Myristic acid CH3-(CH2)12-COOH

16

Palmitic acid CH3-(CH2)14-COOH

18

Stearic acid

20

Arachidic acid CH3-(CH2)18-COOH

H3C H3C H3C

CH3-(CH2)16-COOH

COOH COOH COOH COOH

H3C

COOH

H3C

Examples of unsatturated fatty acids Palmitoleic acid

ω7

16:1,9

Oleic acid

ω9

18:1,9

Linoleic acid

w6

18:2,9,12

H3C

α-Linolenic acid

ω3

18:3,9,12,15

H3C

Arachidonic acid

ω6

20:4,5,8,11,14

H3C

COOH COOH

H3C

COOH COOH

H3C

COOH

The carboxy group of the fatty acids has a pKa value close to 4.8. Only cis-double bonds are present in mammalian metabolism. However, bacteria also produce trans-fatty acids. Since the fatty acids in cows milk were produced by their intestinal bacteria milk fat contains about 4 % trans-fatty acids. Trans double bonds also occur in hydrogenated fats, such as margarine and peanut butter (at about the same concentration as in milk fat). Cis-double bonds decrease the melting points of the fatty acids and triglycerides containing them: O O

H2C O O CH

O

C O P O R H2 O unsaturated fatty acid in 2-position of glycerol

kink at cis-double bond disturbs alignment of molecules -> lower melting point.

Polyunsaturated fatty acids are subject to nonenzymatic auto-oxidation or peroxidation. The process is mediated by free-radical chain reactions. It occurs outside the body, where it causes fats to turn rancid, as well as in the body. Lipid peroxidation can be suppressed by antioxidants such as vitamins A, C and E. Some forms of lipofuscin (“age pigment”) are formed by nonenzymatic reactions involving partially oxidized fatty acids.

362

20.3.1

Adipose tissue

20.2. Utilization of Dietary Fat The products of pancreatic lipase (fatty acids and 2-monoacylglycerol are absorbed. The absorption of long-chain fatty acids (> 16 carbons) requires bile salts. In the mucosal cells, the fatty acids are activated to their CoA-thioesters: The carboxy group of the fatty acids has aPP pKi value close to 4.8. Only cis double bonds are present in naturally occurring fatty acids. Trans double bonds occur in ATP AMP hydrogenated fats, such as margarine and peanut butter. Cis double-bonds decrease the O melting points of the fatty acids and triglycerides containing them. CoA acids are subject to nonenzymatic HS fatty R COO CoA R C Sauto-oxidation Polyunsaturated or Acyl-CoA synthetase peroxidation. The process is mediated by free-radical chain reactions. It occurs outside the body, where it causes fats to turn rancid, as well as in the body. Lipid peroxidation can be suppressed by antioxidants such as vitamins A, C and E. Some forms of lipofuscin (“agemucosal pigment”)cells are synthesize formed by nonenzymatic reactions involving partially oxidized The triglycerides from activated fatty acids andfatty 2-monoglyceride. acids.

+

In the ER, these triglycerides are packaged into chylomicrons, large lipoproteins containing 98–99 % and 1–2 %OF protein. The chylomicrons are released into the lymph and reach II. lipid UTILIZATION DIETARY FAT. the bloodstream via the thoracic duct. Once in the blood, the lifespan of chylomicrons is The products of pancreatic lipase (fatty acids and 2-monoacylglycerol are absorbed. less than 10 min. The absorption of long-chain fatty acids (>16 carbons) requires bile salts. In the mucosal cells, the fatty acids are activated to their CoA-thioesters:

Chylomicron triglycerides areATP hydrolyzed toi fatty acids and 2-monoacylglycerol by lipoproAMP, PP O tein lipaseR (LPL), an enzyme on the lumenal surface of the capillary endothelium. It is COO- + HS CoA R C S CoA present in most tissues but not in brain and liver. Different tissues express different isoenAcyl-CoA synthetase The mucosal cells synthesize triglycerides from tissue, activatedbut fattynot acids and and 2- myocardium, zyme forms of LPL. The LPL activity in adipose muscle monoglyceride. In the ER, these triglycerides are packaged into chylomicrons, large islipoproteins increasedcontaining by insulin. This directs dietary fat to adipose tissue in the well-fed state. Treat98 – 99% lipid and 1-2% protein. The chylomicrons are released ment of lymph patients releasesviaLPL into the circulation. into the and with reach heparin the bloodstream the thoracic duct. Once in the blood, the lifespan of chylomicrons is less than 10 minutes. Chylomicron triglycerides are hydrolyzed to fatty acids and 2-monoacylglycerol by lipoprotein lipase (LPL), an enzyme on the lumenal surface of the capillary endothelium. It is present in most tissues but not in brian and liver. Different tissues express different isoenzyme forms of LPL. The LPL activity in adipose tissue, but not muscle and myocardium, is increased by insulin. This directs dietary fat to adipose tissue in the wellfed state. Treatment of patients with heparin releases LPL into the circulation.

20.3. Adipose tissue The

III. Adipose tissue The pathway of fat synthesisin in adipose differs from from that in that the intestine: pathway of fat synthesis adiposetissue tissue differs in the

Glucose 2 Acyl-CoA Phosphatidic acid Pi

intestine:

Dihydroxyacetone Phosphate NADH Glycerol phosphatedehydrogenase NAD+ Glycerol 3-phosphate

Acyl-CoA Triglyceride

164

363

20.4

Biochemistry and Genetics

20.3.1. Triglyceride Glycerol phosphate, which has to be made from glucose, is an important limiting factor. Therefore fat synthesis is stimulated by insulin, which is required for glucose transport into adipose cells. Most of the acyl-CoA comes from triglycerides in chylomicrons and VLDL. Lipolysis (fat hydrolysis) is initiated by hormone-sensitive adipose tissue lipase. This enzyme is activated by cAMP-dependent phosphorylation. Norepinephrine and epinephrine raise cAMP in adipose tissue by an action on β-receptors. Thyroid hormone, glucocorticoids and growth hormone promote lipolysis by inducing the synthesis of proteins involved in cAMP-responsiveness. Insulin inhibits the hormone-sensitive lipase. Therefore phosphate, which has to be made from an important limiting fat is Glycerol degraded during fasting (low insulin) andglucose, during isphysical exercise and stress (high factor. Therefore fat synthesis is stimulated by insulin, which is required for glucose norepinephrine and epinephrine). transport into adipose cells. Most of the acyl-CoA comes from triglycerides in chylomicrons and VLDL. The fatty acids during lipolysis are released intoadipose the blood. Lipolysis (fatformed hydrolysis) is initiated by hormone-sensitive tissueThey lipase.are transported This enzyme is activated by cAMP-dependent phosphorylation. Norepinephrine and acids levels are to other tissues non-covalently bound to serum albumin. Plasma free fatty epinephrine raise cAMP in adipose tissue by an action on β-receptors. Thyroid hormone, lowest after a carbohydrate meal and highest in long term fasting. glucocorticoids and growth hormone promote lipolysis by inducing the synthesis of proteins involved in cAMP-responsiveness. Insulin inhibits the hormone-sensitive lipase. Therefore fat is degraded during fasting (low insulin) and during physical exercise and stress (high norepinephrine and epinephrine). The fatty acids formed during lipolysis are released into the blood. They are transported to other tissues noncovalently bound to serum albumin. Plasma free fattty acids levels are lowest after a carbohydrate meal and highest in long term fasting.

20.4. Fatty Acid Oxidation

With the exception of specialized cells such as neurons and RBCs, all cells can oxidize fatty IV. Fatty Acid Oxidation. acids. The fatty acids are either albumin-bound “free” (unesterified) fatty acids, or they are derived triglycerides in lipoproteins. use of lipoprotein depends on Withfrom the exception of specialized cells suchThe as neurons and RBCs, alltriglycerides cells can oxidize fatty acids. The fatty acids are either albumin-bound “free” (unesterified) fatty acids, LPL, and free fatty acids are used in proportion to their plasma concentration. or they are derived from triglycerides in lipoproteins. The use of lipoprotein triglycerides depends on LPL, and free fatty acids are used in proportion to their plasma concentration. Oxidation takes place in the mitochondria. Long-chain fatty acids (> 14 carbons) are acOxidation takes place in the mitochondria. Long-chain fatty acids (>14 carbons) are tivated as as CoA-thioesters CoA-thioesters in cytoplasm the cytoplasm theninto shuttled into the mitochondrion as activated in the and thenand shuttled the mitochondrion as acyl-carnitine. acyl-carnitine: Mitochondrion

Cytoplasm Carnitine Acyl-CoA CAT-1 CoA-SH Acyl-carnitine

Carnitine Acyl-CoA CAT-2 CoA-SH Acyl-carnitine

The carnitine shuttle is rate-limiting for β-oxidation. Carnitine-acyl-transferase I (CAT-I) is inhibited by malonyl-CoA, an intermediate of fatty acid synthesis. The carnitine shuttle is rate-limiting for β-oxidation. Carnitine-acyl-transferase In β-oxidation, C-3 (the β-carbon) of the CoA-activated fatty acid is oxidized, and the is inhibited by malonyl-CoA, an intermediate of carbons fatty acid synthesis. fatty acid shortened in 2 carbon decrements. These two are released as acetylCoA. One FADH2 and one NADH are produced in each cycle (see next pg):

I (CAT-I)

O In β-oxidation, C-3 FAD (theFADH β-carbon)Oof the HCoA-activated fatty acid is oxidized, and the fatty OH O O R two C C carbons C S CoA are released as acetyl-CoA. One R C C C S CoA acid shortened 2 carbon decrements. These R C C C in S CoA H H H H 2

H2 H2

Alcohol Dehydrogenase

2

2

O

2

2

Acetyl-CoA

R C S CoA

364

CoA-SH

OH

NAD

NADH, H+ O

R C C C S CoA H H2

Energy yield from β-oxidation of one palmitic acid: 165

Fatty Acid Oxidation

20.4

FADH2 and one NADH + H+ are produced in each cycle: O Carnitine Acyl-carnitine

CoA SH

Carnitine acyl transferase II

Carnitine O S CoA Acyl-CoA

FAD

Acyl-CoA dehydrogenase

FADH2 O S CoA trans-∆2-enoyl-CoA

Enoyl-CoA hydratase

H2O

OH

O S CoA

L-β-hydroxyacyl-CoA +

NAD Hydroxyacyl-CoA dehydrogenase

NADH + H+ O

O S CoA

β-ketoacyl-CoA

CoA-SH Thiolase

O

O

S CoA Acyl-CoA (-2 C)

+

S CoA Acetyl-CoA

Energy yield from β-oxidation of one palmitic acid:

365

20.6

Biochemistry and Genetics

Metabolite equivalent to 7 FADH 14 ATP 7 NADH 21 ATP 7 FADH ------------------------> 14 ATP 8 acetyl CoA------------------------96 ATP 7 NADH > 21 ATP 8 acetylof CoA ------------------> 96 ATP Activation fatty acid -2 ATP Activation of fatty acid ----------------> -2 ATP 7 FADH ------------------------> 14 ATP 129 ATP 129 ATP 7 NADH ------------------------Efficiency of ATP synthesis: 40 %. > 21 ATP 8 acetyl CoA ------------------- > 96 ATP Efficiency of ATP synthesis: 40%. Activation of fatty acid ----------------> -2into ATP Unsaturated fatty acids are channeled β-oxidation by specialized Unsaturated fatty acids are channeled into β-oxidation by specialized enzyme enzyme systems. OddATP fatty acids propionyl-CoA remaining at the at endthe of end β-oxidation. Propionylsystems.chain Odd-chain fattyleave acidsone leave one 129 propionyl-CoA remaining of βCoA channeled channeled into the TCA cycle: oxidation. Propionyl-CoA into the TCA cycle: COsynthesis: Efficiency of ATP 40%. 2 Methylmalonyl-CoA Propionyl CoA fatty acids are Succinyl-CoA Unsaturated channeled into β-oxidation by specialized enzyme Propionyl-CoA Methylmalonyl-CoA systems. Odd-chain fatty acids leave one propionyl-CoA remaining at the end of βcarboxylase mutase (B12) oxidation. Propionyl-CoA channeled into the TCA cycle: (biotin) CO2 Methylmalonyl-CoA Propionyl CoA Succinyl-CoA Branched-chain acids require a specialized pathway called α-oxidation. Branched-chainfatty fatty acids require a specialized pathway called α-oxidation. Very long fatty Propionyl-CoA acids (>20 carbons) require peroxisomal β-oxidation. Very long fatty Methylmalonyl-CoA acids accumulate in carboxylase patients with syndrome, a rare,(Brecessively inherited mutase 12) Very long fatty acids (> 20 Zellweger carbons) require peroxisomal β-oxidation. Very long fatty acids absence of peroxisomes. (biotin)

accumulate in patients with Zellweger syndrome, a rare, recessively inherited absence of V.peroxisomes. Ketogenesis. Branched-chain fatty acids require a specialized pathway called α-oxidation. “Ketone bodies” include acetoacetate, β-hydroxybutyrate and acetone. Acetoacetate long fatty acids carbons)products require peroxisomal and Very β-hydroxybutyrate are (>20 water-soluble formed from β-oxidation. acetyl-CoA inVery liverlong fatty acids accumulate in patients with Zellweger syndrome, a rare, recessively mitochondria. They are released into the blood for transport to other tissues where they can inherited absence of20.5. peroxisomes. Ketogenesis be oxidized. Acetone is formed nonenzymatically from acetoacetate. It has no known biological function and is exhaled through the lungs. Large amounts of ketone bodies are formed long-term fasting and in diabetics. Biosynthetic pathway: V. during Ketogenesis.

CoA Acetyl-CoA CoA “Ketone bodies” include acetoacetate, β-hydroxybutyrate and acetone. and β“Ketone bodies” include acetoacetate, β-hydroxybutyrate and acetone.Acetoacetate Acetoacetate 2 Acetyl-CoA hydroxybutyrate are water-soluble products formed from acetyl-CoA in liver mitochondria. Acetoacetyl-CoA HMG-CoA and β-hydroxybutyrate are water-soluble products formed from acetyl-CoA in liver TheyThey are released into the for transport to other tissuestissues where where they can be can oxidized. mitochondria. are released intoblood the blood for transport to other they Acetyl-CoA Acetone is formed nonenzymatically from acetoacetate. It has no known function be oxidized. Acetone is formed nonenzymatically from acetoacetate. It hasbiological no known Acetoacetate β-Hydroxybutyrate is exhaled thethrough lungs. Large amounts of ketone bodies are formed during biological and function and isthrough exhaled the lungs. Large amounts of ketone bodies are long+ NAD NADH, H+ termlong-term fasting and in diabetics. Biosynthetic pathway: formed during fasting and in diabetics. Biosynthetic pathway:

CoA

Acetyl-CoA

CoA

Extrahepatic tissues utilize ketone bodies by converting acetoacetate to acetoacetylCoA.

2 Acetyl-CoA

Acetoacetyl-CoA

HMG-CoA

VI. Aberrations Of Fatty Acid Metabolism. β-Hydroxybutyrate

Acetyl-CoA Acetoacetate

Essential fatty acid deficiency is seen only in patients on total parenteral nutrition or with severe fat malabsorption. NAD+ NADH, H+

CoA.

Extrahepatic tissues utilize ketone bodies by converting acetoacetate to acetoacetylExtrahepatic tissues utilize ketone bodies by converting acetoacetate to acetoacetyl-CoA. 166

VI. Aberrations Of Fatty Acid Metabolism. Essential fatty acid deficiency is seen only in patients on total parenteral nutrition 366 or with severe fat malabsorption.

166

From Carbohydrate To Fat

20.7

20.6. Aberrations Of Fatty Acid Metabolism Essential fatty acid deficiency is seen only in patients on total parenteral nutrition or with severe fat malabsorption. Defects of mitochondrial β-oxidation can affect muscle or liver. If muscle is affected: muscle weakness, cramping, myoglobinuria, and fat accumulation in the cytoplasm. If the liver is affected: Hypoketotic hypoglycemia during fasting. Although most fatty acids are not substrates of gluconeogenesis, fatty acid oxidation supplies the energy for gluconeogenesis. β-oxidation is impaired in: Carnitine deficiency: Either generalized or affecting only muscle. Responds to exogenous carnitine, or to a diet with medium-chain instead of long-chain fatty acids. Carnitine-acyl transferase deficiency: Similar to carnitine deficiency, usually limited to muscle. Medium-chain acyl-CoA dehydrogenase deficiency: Both liver and muscle are affected. Hypoketotic hypoglycemia develops during fasting. Refsum’s disease is a rare inherited deficiency of α-oxidation. It leads to an accumulation of phytanic acid, a branched-chain fatty acid from green vegetables and ruminant fat.

20.7. From Carbohydrate To Fat Excess carbohydrate and protein can be converted to fat in the liver. Acetyl-CoA (from carbohydrate or protein) is turned into fatty acids, and the fatty acids are esterified into triglycerides. These triglycerides are secreted in VLDL for use by other tissues. The immediate substrate for fatty acid biosynthesis is malonyl-CoA, synthesized from acetyl-CoA by the cytoplasmic acetyl-CoA carboxylase: ADP HCO3 P H2O ATP i

O H3C C S CoA

O -

Acetyl-CoA carboxylase (Biotin)

OOC C C S CoA H2

In addition to malonyl-CoA, fatty acid synthesis requires NADPH + H+ for the reductive reactions. The cytoplasmic fatty acid synthase complex contains two important SH−groups to which the growing fatty acid and the incoming malonyl group are bound covalently. One is in a

367

20.8

Biochemistry and Genetics

cysteine side chain, the other in phosphopantetheine, a covalently bound prosthetic group. In each cycle, 2 carbons (from malonyl-CoA) are added to the fatty acid. Reductive reactions occur while the growing chain os bound to phosphopantetheine. Palmitate (saturated, 16 carbons) is the principal product. Overall reaction: Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + H+ → Palmitate + 8 CoA-SH + 14 NADP+ + 7 CO2 ATP is required only for the synthesis of malonyl-CoA. The acetyl-CoA for fatty acid synthesis is shuttled from the mitochondrion in the form of citrate. In the cytoplasm, citrate is cleaved by ATP-citrate lyase to oxaloacetate and acetyl-CoA. Oxaloacetate is shuttled back into the mitochondrion in a reaction sequence in which malic enzyme (reaction: malate → pyruvate) produces NADPH + H+ . Acetyl-CoA carboxylase, fatty acid synthase, ATP-citrate lyase, and glucose-6-phosphate dehydrogenase in the liver are induced by carbohydrate feeding and insulin. Acetyl-CoA carboxylase is allosterically stimulated by citrate and inhibited by acyl-CoA. Citrate is high after a carbohydrate meal, and acyl-CoA is high during fasting and after a fat meal. The enzyme is also inactivated by cAMP-dependent phosphorylation and activated by insulin-stimulated dephosphorylation. Other fatty acids are synthesized from palmitate: chain-elongation produces fatty acids with up to 24 or 26 carbons. Desaturation is initiated in position ∆9 , and further double bonds are introduced between this first double bond and the carboxy group. The liver synthesizes triglycerides and forms VLDL both in the fed state, using newly synthesized fatty acids, and during fasting using fatty acids from adipose tissue.

20.8. Phosphoglyceride Metabolism Phosohoglycerides are synthesized from phosphatidic acid. Phosphatidic acid is “activated” to CDP-diacylglycerol, and this reacts with alcohol. Used for the synthesis of phosphatidylinositol. Phosphoglycerides can also be synthesized from 1,2-diacylglycerol and a CDP-activated alcohol. Used for phosphatidylethanolamine and phosphatidylcholine. Phospholipases are required for the remodeling and catabolism of the phosphoglycerides. They are grouped according to their cleavage specificities:

368

Phosohoglycerides are synthesized from phosphatidic acid. Phosphatidic acid is “activated” to CDP-diacylglycerol, and this reacts with alcohol. Used for the synthesis of phosphatidylinositol. Phosphoglycerides can also be synthesized from 1, 2 –diacylglycerol and a CDPSphingolipid Metabolismand phosphatidylcholine. 20.9 activated alcohol. Used for phosphatidylethanolamine Phospholipases are required for the remodeling and catabolism of the phosphoglycerides. They are grouped according to their cleavage specificities: A1

B

B

A1

O

O

O C R1

C O H2C O CH2R 1

O O R2

A2

H2C O OC RH1 C 2

C O CH R2

CO O CH

C O P O R3 C O H2 H2 O

A2

P O R3 O

D

C

HO CH

O

HO CH

O C R1 O

C O P O C RO3 P O H2 H2 O O

R3

D

C

Fatty acids can be replaced by the sequential of a phospholipase and aselechighly Fatty acids can be replaced by the sequential action ofaction a phospholipase and a highly selective acyl-transferase tive acyl-transferase CO2 Phosphatidylserine

3 SAM Phosphatidylethanolamine

Phosphatidylethanolamine + Serine

Phosphatidylcholine

Phosphatidylserine + Ethanolamine

20.9. Sphingolipid Metabolism The sphingolipid are synthesized from ceramide (= sphingosine + fatty acid). Synthesis 168 sugars are used for the synthesis of the takes place in the ER, where nucleotide-activated glycosphingolipids. Glycolipids are degraded by highly specific lysosomal exoglycosidases (+ sulfatases). A recessively inherited deficiency in any of the glycolipid-degrading enzymes results in a sphingolipidosis (= lipid storage disease), with an accumulation of glycolipid substrate in lysosomes. Neurological deterioration and hepatosplenomegaly are frequent clinical signs: Tay-Sachs disease: Deficiency of hexosaminidase A, with accumulation of ganglioside GM2. Hepatosplenomegaly, neurological deterioration, blindness (“amaurotic idiocy”), cherry-red spot on the macula of the eye, death at about 3 years. Most common in Ashkenazi Jews. Gaucher’s disease: Deficiency of glucocerebrosidase. Glucocerebroside accumulates. The rare infantile form is similar to Tay-Sachs. The more common adult-onset form (mostly in Ashkenazi Jews) presents with splenomegaly, thrombocytopenia, abdominal problems and bone erosion presenting in midlife, but no mental deficiency.

369

substrate in lysosomes. Neurological deterioration and hepatosplenomegaly are frequent clinical signs. Tay-Sachs disease: Deficiency of hexosaminidase A, with accumulation of ganglioside GM2. Hepatosplenomegaly, neurological deterioration, blindness (“amaurotic 20.10 cherry-red spot on the macula Biochemistry and death Genetics idiocy”), of the eye, at about 3 years. Most common in Ashkenazi Jews. Gaucher’s disease: Deficiency of glucocerebrosidase. Glucocerebroside Other lipid storage diseases include metachromatic leukodystrophy, accumulates. The rare infantile form Krabbe’s is similar todisease, Tay-Sachs. The more common adultNiemann-Pick disease. onset form (mostly in Ashkenazi Jews) presents with splenomegaly, thrombocytopenia, abdominal problems and bone erosion presenting in midlife, but no mental deficiency. Other lipid storage diseases include Krabbe’s disease, metachromatic leukodystrophy, Niemann-Pick disease.

20.10. Cholesterol And Bile Acids X.

CHOLESTEROL AND BILE ACIDS

Cholesterol is both issynthesized endogenously and derived animal sources. Cholesterol both synthesized endogenously and from derived from dietary animal dietary Vegan diets are essentially cholesterol-free. Intestinal absorption is about 50 %. The liver sources. Vegetarian diets are essentially cholesterol-free. Intestinal absorption is about accounts for 50 % of the endogenous synthesis. Endocrine glands which produce steroid 50%. The liver accounts for 50% of the endogenous synthesis. Endocrine glands which produce steroid hormones have high synthesis. rates of cholesterol synthesis. typical% hormones also have high ratesalso of cholesterol On a typical AmericanOn diet,a 50–65 American diet, 50 – 65% of body cholesterol is from endogenous synthesis. of body cholesterol is from endogenous synthesis. Acetyl-CoA (C-2)

HMG-CoA (C-6)

Mevalonate (C-6)

Cholesterol (C-27)

Lanosterol (C-30)

Squalene (C-30)

Isopentenyl-pyrophosphate (C-5) Dimethylallyl-pyrophosphate (C-5)

The first reactions, up to HMG-CoA, are shared with the pathway of ketogenesis.

The reactions, up to HMG-CoA, are shared with the ispathway of ketogenesis. But But first ketogenesis occurs in the mitochondria, while cholesterol synthesized in Cytosol and ketogenesis occurs in the mitochondria, while cholesterol is synthesized in Cytosol and ER. ER. The 5-carbon intermediates isopentenyl-pyrophosphate and DimethylallylThe 5-carbon intermediates isopentenyl-pyrophosphate and dimethylallyl-pyrophosphate pyrophosphate are the building blocks of the isoprenoids, which include ubiquinone, lipid groups involvedblocks in anchoring membrane proteins, and many plant products. The reductive are the building of the isoprenoids, which include ubiquinone, lipid groups involved in the pathway require NADPH. insteps anchoring membrane proteins, and many plant products. The reductive steps in the +. reductase ---! mevalonate) catalyzes the committed step of pathwayHMG-CoA require NADPH + H(HMG-CoA cholesterol biosynthesis. Free cholesterol reduces the amount and activity of the enzyme. Fasting reduces and insulin increases its activity. catalyzes the committed step of cholesHMG-CoA reductase (HMG-CoA → mevalonate)

terol biosynthesis. Free cholesterol reduces the amount and activity of the enzyme. Fasting reduces and insulin increases its activity.

169 in the body. Cholesterol is excreted in the The steroid nucleus of cholesterol is not degraded bile as free cholesterol or after its conversion to bile acids. The committed step of bile acid synthesis is the introduction of a hydroxy group by 7α-hydroxylase in the ER. This enzyme is feedback-inhibited by bile acids. The primary bile acids cholic acid and chenodeoxycholic acid are secreted as conjugates with glycerine or taurine.

In the gut, the primary bile acids are de-conjugated and reduced to the secondary bile acids deoxycholic acid and lithocholic acid by intestinal bacteria. 98 % of the bile acids are absorbed in the ileum. Then picked up by the liver and re-secreted into the bile. This is called entero-hepatic circulation. An average bile acid molecule is recycled 5–8 times per day and persists for about a week in the entero-hepatic system.

370

Lipoprotein Composition

20.11

The steroid nucleus of cholesterol is not degraded in the body. Cholesterol is excreted in the bile ascholesterol free cholesterol its conversion to bile The committed Biliary has or to after be kept in solution as acids. a component of mixed micelles. Most step of bile acid synthesis is the introduction of a hydroxy group when by 7α-the hydroxylase gallstones consist of cholesterol. They form cholesterolin the content of the bile is ER. This enzyme is feedback-inhibited by bile acids. The primary bile acids cholic acid increased or the concentration of bile salts or phospholipid is reduced. Gallstones are most and chenodeoxycholic acid are secreted as conjugates with glycerine or taurine. common in fat, fertile females. In the gut, the primary bile acids are de-conjugated and reduced to the secondary bile acids deoxycholic acid and lithocholic acid by intestinal bacteria. 98% of the bile acids are absorbed in the ileum. Then picked up by the liver and re-secreted into the bile. This is called entero-hepatic circulation. An average bile acid molecule is recycled 5-8 times per day and persists for about a week in the enterohepatic system. Biliary cholesterol has to be kept in solution as a component of mixed micelles. 20.11. Composition Most gallstones consistLipoprotein of cholesterol. They form when the cholesterol content of the bile is increased or the concentration of bile salts or phospholipid is reduced. Gallstones are most common in fat, fertile females. XI.

LIPOPROTEIN COMPOSITON

Typical fasting lipid concentrations are: Normal TypicalLipid fasting lipid concentrations are:Range (mg/dL) Total lipid 400–800 LipidTriglycerides Normal Range (mg/dL) 40–300 Total cholesterol 120–280 Total lipid 400 – 800 Cholesterol esters 90–200 Triglycerides 40 – 300 Phospholipids 150–380 Total cholesterol 120 - 280 Cholesterol esters 90 – 200 Free fatty acids 8–14 Phospholipids Free fatty acids

150 - 380 8 - 14

With the exception of free fatty acids (from adipose tissue), lipids are present thepresent in the blood With the exception of free fatty acids (from adipose tissue), lipids inare blood as lipoproteins. General structure of lipoproteins. as lipoproteins. General structure of lipoproteins: = Cholesterol = Cholesterol-ester

Lipoprotein

= Phospholipid = Triglyceride

Hydrophobic lipids (triglycerides, cholesterol esters) are center in the of center Hydrophobic lipids (triglycerides, cholesterol esters) are in the the of the lipoprotein lipoprotein particle, and amphipathic lipids, mainly phospholipids, form a water/lipid particle, and amphipatic lipids, mainly phospholipids, form a water/lipid interface. Also the interface. Also the apolipoproteins are amphipathic. side ofisthe protein is (facing triglyceride apolipoproteins are amphipathic. One side of One the protein hydrophobic hydrophobic (facing triglyceride core) and the other side is hydrophilic. core) and the other side is hydrophilic.

170 Lipoproteins are classified according to their density: protein-rich (proteins ρ ≈ 1.5 mg/ml) particles are more dense than lipid-rich (lipids ρ ≈ 0.9 mg/ml) particles. They can also be separated by electrophoresis at pH 8.6:

371

Lipoproteins are classified according to their density: 20.12 Biochemistry andprotein-rich Genetics particles are more dense than lipid-rich particles. They can also be separated by electrophoresis at pH 8.6: Density range (g/cm3)

Electrophoretic result at pH 8.6 Origin

Chylomicrons (no migration)

< 0.95

β-lipoproteins pre-β-lipoproteins

1.006-1.063 0.95 - 1.006

α-Lipoproteins

= LDL = VLDL = HDL

1.063-1.21

+ Properties and composition of plasma lipoproteins: Lipid percent by class Lipoprotein PropertiesClass and

Diameter of Protein Triglyc. Phosphol Cholesterol composition plasma lipoproteins: Lipoprotein Class (nm) Diameter Protein(%) Triglyc.ester Phosphol free Chol. (%) (%) (%) free (%) Chylomicron 100-1000(nm) 1-2 86(%) 8 2 (%)3 (%) (%) Very-low-density 30-80 100–1000 6-10 551–2 18 78 Chylomicron 8613 3 Intermediate density 25-30 15-20 25 21 28 9 Very-low-density 5540 18 13 Low density 20-25 30–80 22 9 6–10 20 8 High density (HDL-2) 9-12 35-45 5 15–20 33 5 Intermediate density 25–30 2517 21 28 High density (HDL-3) 5-9 50-55 3 22 28 3 Low density 20–25 912 20 40 HighWith density (HDL-2) 9–12 (LDL, 35–45 5 (chylomicrons), 33 the 17 the exception of apoB-100 VLDL) and apoB-48 apolipoproteins readily exchange5–9 between different lipoprotein particles. Apolipoproteins High density (HDL-3) 50–55 3 28 12 regulate enzymes of lipoprotein metabolism, mediate the cellular uptake of lipoprotein particles, and transfer lipids between different lipoprotein classes and between lipoproteins and cells.

Chol. ester (%) 2 7 9 8 5 3

With the exception (LDL, Lipoprotein VLDL) and apoB-48 (chylomicrons), the apolipoproApoprotein Molecularof WtapoB-100 Plasma conc Functions (mg/ml) different components teins readily(kiloDaltons) exchange between lipoprotein particles. Apolipoproteins regulate enA-I 29 130 HDL, chylomics. Activates LCAT zymes of lipoprotein metabolism, mediate the cellularUnknown uptake of lipoprotein particles, and A-II 17 40 HDL, chylomics. transfer lipids classes andPrimarily between lipoproteins and cells. B-48 241 between different variable lipoprotein chylomicrons structural B-100 513 Molecular 80Wt Plasma VLDL, LDL Lipoprotein LDL uptake by cells Apo-protein conc comFunctions C-I 7 6 All except LDL unknown ponents C-II 9 3 All except LDL Activates LPL (kDa) (mg/ml) C-III 9 12 All except LDL Inactivates LPL D A-I 19 10 29 HDL130 HDL, chylomics. unknown Activates LCAT E 34 5 VLDL, IDL, remnant uptake by A-II 17 40 HDL, chylomics. chylomics. liver B/E receptorsUnknown B-48

241

B-100

513

80

VLDL, LDL

C-I C-II C-III D E

7 9 9 19 34

6 3 12 10 5

All except LDL All except LDL All except LDL HDL VLDL, IDL, chylomics.

372

variable

171

chylomicrons

Primarily structural LDL uptake by cells unknown Activates LPL Inactivates LPL unknown remnant uptake by liver B/E receptors

Lipoprotein Metabolism

20.12

20.12. Lipoprotein Metabolism There are three highways of lipoprotein-based lipid transport: 1. Dietary lipids from the intestine to other tissues. The vehicles are chylomicrons and chylomicron remnants. 2. Lipids from endogenous synthesis in the liver to other tissues. The vehicles are VLDL and LDL. 3. Reverse cholesterol transport to the liver. The initial vehicle is HDL. Unlike the other lipids, cholesterol cannot be degraded in extrahepatic tissues. It has to be transported to the liver for biliary excretion or conversion to bile acids. Chylomicrons are converted to chylomicron remnants by lipoprotein lipase (LPL) in adipose tissue, muscle and elsewhere. These remnants are endocytosed by the liver. Endocytosis requires apoE, which binds to a receptor on the surface of hepatocytes in the space of Disse. The liver synthesizes 25–50 g of triglyceride per day (+ cholesterol and phospholipid). These lipids are released into the circulation as very-low density lipoproteins (VLDL). VLDL is released with apoB-100 as its major apolipoprotein. Most other apolipoproteins are acquired from HDL in the circulation. Like chylomicrons, VLDL is initially metabolized in peripheral tissues by LPL. In the blood, VLDL triglycerides have half-lives of 15–60 min (chylomicrons: 5–10 min). Like chylomicron remnants, VLDL remnants possess apoE, and about half of them are endocytosed through liver apoE receptors. Smaller remnants which appear in the blood as intermediate-density lipoproteins (IDL) are processed to low-density lipoproteins (LDL). This involves the action of hepatic lipase as well as the transfer of excess apolipoproteins to HDL. LDL has a solitary apoB-100 molecule which mediates endocytosis through the LDL receptor in liver (60 %) and extrahepatic tissue (40 %). LDL is the major source of external cholesterol for extrahepatic tissues. Alternative receptors, known as scavenger receptors, mediate LDL uptake in macrophages. Most cells obtain cholesterol both from endogenous synthesis and endocytosed LDL. Free cholesterol in the cell regulates three important proteins: • Acyl-CoA-cholesterol-acyl transferase (ACAT) is induced resulting in increased formation of cholesterol esters for storage. • HMG-CoA reductase is repressed, resulting in reduced cholesterol synthesis. • LDL receptors are down-regulated (decreased receptor synthesis). This raises the level of circulating LDL.

373

20.14

Biochemistry and Genetics

High-density lipoprotein (HDL) occurs in several subtypes which represent different stages of its metabolism. Nascent HDL, released by the liver, is a small, phospholipid-rich particle resembling a disk of lipid bilayer associated with apolipoprotein. Once in the circulation, nascent HDL acquires unesterified cholesterol from other lipoproteins and cells. The acquisition of cholesterol from cells requires the transient binding of HDL to cell surfaces. Binding and subsequent cholesterol transfer requires apoA-I, the major apolipoprotein of HDL, and/or apoE. HDL-cholesterol becomes esterified by lecithin-cholesterol acyl transferase (LCAT), which transfers a fatty acid from lecithin to cholesterol. Most of the cholesterol esters are then transferred from HDL to triglyceride-rich lipoproteins and remnant particles by the cholesterol ester transfer protein (CETP), much of this in exchange for triglycerides. The remnants bring the cholesterol esters to the liver. Some cholesterol also reaches the liver by direct transfer from HDL or by the endocytosis of apoE-coated HDL particles.

20.13. Lipoproteins and Atherosclerosis The fatty streak consists of cholesterol ester deposits in the intima of large arteries. Macrophages take up LDL through their scavenger receptors. They prefer chemically modified LDL, for example oxidatively damaged LDL. These receptors may be important for the removal of aberrant or aged lipoproteins that are no longer good ligands for other lipoprotein receptors. The uptake of LDL-cholesterol has to be balanced by the transfer of excess cholesterol from macrophages to HDL. A foam cell develops only when the amount of cholesterol acquired from LDL exceeds the amount released to HDL. When the foam cell dies, the intracellular lipid droplets become extracellular. An atheromatous plaque develops when intimal smooth muscle cells proliferate, probably in response to the lipid deposits and to locally-released growth factors and cytokines. Early lesions are asymptomatic, but the complications of advanced atherosclerosis (heart attack, stroke) are responsible for 30 % of all deaths in the US. The risk factors for atherosclerosis include age and gender (lesions progress with age, and males are affected earlier than females), smoking, diabetes mellitus, and hypercholesterolemia. A low HDL/LDL ratio predicts a high risk of atherosclerotic lesions, while a high HDL/LDL ratio is protective. The HDL/LDL ratio is protective. The HDL/LDL ratio can be increased by exercise, low caloric intake, a high dietary unsaturated/saturated fat ratio, low dietary cholesterol, high dietary fiber, regular alcohol consumption, and some drugs including cholesterol synthesis inhibitors cholestyramine, and niacin.

374

Lipoprotein Disorders

20.14

20.14. Lipoprotein Disorders Abetalipoproteinemia, a rare recessive disease, is caused by the absence of a triglyceride transfer protein in the ER. As a result, liver and intestine are unable to synthesize and secrete apoB-rich lipoproteins. VLDL, LDL and chylomicrons are essentially absent. This disease leads to severe fat malabsorption and signs of vitamin E deficiency with abnormalities of RBCs, CNS and muscle. Tangier disease is a rare recessive disease in which HDL levels are reduced to less than 5 % of normal. There is a marked deficiency of apoA-I, the major apolipoprotein of HDL. LDL is also reduced. There is only a mild tendency for early atherosclerosis, probably because decreased LDL cholesterol reduces the need for reverse cholesterol transport by HDL. The molecular cause for Tangier-disease is a defect in the ABCA1 gene, which encodes for an ABC-type membrane transporter which transfers phosphatidyl choline (PC) from the plasma membrane to apoA-I. The disease is named after an island in the Chesapeake Bay, where it was discovered. Familial hypercholesterolemia is a dominantly inherited condition (incidence 1 in 500) caused by a deficiency of LDL receptors in liver and peripheral tissues. LDL accumulates to twice normal levels and total cholesterol is increased to 250–500 mg/dL. Xanthomas (visible subcutaneous lipid deposits) develop in most patients by age 20 a. Early death by coronary disease is common. CETP deficiency is a benign condition in which cholesterol esters (formed by LCAT) cannot be transferred from HDL to other lipoproteins. HDL-cholesterol is elevated about four-fold. Cholesterol reverse transport is still accomplished by endocytosis of apoEcontaining HDL particles or direct transfer of cholesterol esters to the liver. This trait is common in Japan. Hyperlipoproteinemias are grouped into five types. These are not diseases but phenotypes which occur in a variety of contexts. Most are “multifactorial”, but some are singlegene or are secondary to a chronic disease such as diabetes mellitus, alcoholism or hypothyroidism. Type I hyperlipoproteinemia, or hyperchylomicronemia, is a rare form in which chylomicron hydrolysis is impaired. Some patients have an inherited deficiency of lipoprotein lipase (LPL), others are lacking apoC-II, an obligatory activator or LPL. The patients have massive hypertriglyceridemia, but not much atherosclerosis. LDL is reduced. The patients have to avoid dietary fat. Type II hyperlipoproteinemia, or hypercholesterolemia, is an elevation of LDL. Multifactorial and secondary forms are far more common than familial hypercholesterolemia. This pattern is a common risk factor for atherosclerosis and coronary heart disease.

375

20.15

Biochemistry and Genetics

Type III hyperlipoproteinemia, or dysbetalipoproteinemia, is caused by homozygosity for an apoE variant that is not recognized by hepatic apoE receptors. Chylomicron remnants and LDL-like VLDL remnants accumulate. This pattern is rare. The atherosclerosis risk is increased. Type IV hyperlipoproteinemia, or hypertriglyceridemia, is an elevation of VLDL. This common pattern is associated with diabetes mellitus, alcoholism, excess dietary carbohydrate, progesterone-rich contraceptives, and obesity. Cholesterol is also mildly elevated, and the atherosclerosis risk is increased. Type V hyperlipoproteinemia is rare pattern with a combined elevation of VLDL and chylomicrons, secondary to uncontrolled diabetes mellitus or of unknown etiology. Familial combined hyperlipoproteinemia may be caused by a dominantly inherited gene which causes increased apoB-100 synthesis in the liver. Either the type II or the type IV pattern is expressed. About 1 % of the population are thought to have this disorder.

20.15. Practice Questions Plasma free fatty acid levels are variable. Which organ produces these fatty acids, and under what conditions do you expect elevated levels? Compare the fates of blood-derived free fatty acids in skeletal muscle and the liver during fasting. Do you know of any inborn errors of metabolism that lead to muscle weakness and muscle cramps on exertion? The Ackee fruit, which grows in Jamaica, contains a toxin that inhibits α-oxidation. What are the metabolic consequences, and under what conditions can this toxin be fatal? You start a job at a pharmaceutical company that wants to develop new anti-obesity drugs. In the first staff meeting you are asked if any of the following drug types would be promising, and if undesirable side effects have to be expected: • Inhibitors of pancreatic lipase • Inhibitors of lipoprotein lipase • Inhibitors of hormone-sensitive lipase • β-adrenergic receptor blockers • Inhibitors of protein kinase A

376

Objectives In Summary

20.16

• Inhibitors of cAMP-degrading phosphodiesterases • Inhibitors of glucose transport in adipose tissue

20.16. Objectives In Summary 1. Define the terms “saturated”, “monounsaturated” and “polyunsaturated fatty acid, and relate the physicochemical properties of fatty acids and triglycerides to their degree of unsaturation. 2. Describe the formation of chylomicrons in the intestinal mucosa, and know the importance of lipoprotein lipase for tissue utilization of dietary triglyceride and the changes in its activity in different physiological states. 3. Identify the substrates for triglyceride synthesis in adipose tissue, and the hormonal and nutritional factors that determine their availability. 4. List the most important hormonal effects on hormone sensitive adipose tissue lipase. 5. Describe the role of carnitine for the mitochondrial uptake of long-chain fatty acids. 6. Describe the sequence of reactions and the cofactors for β-oxidation, and identify the tissues that use β-oxidation as an important energy source and the effect of disruptions in this pathway on the functions of these tissues. 7. Identify the tissue and organelle of ketogenesis, and the conditions that favor ketogenesis. 8. Identify the roles of acetyl-CoA carboxylase and the fatty acid synthase complex for fatty acid biosynthesis, and the coenzyme and energy requirements. 9. List the influences of hormones and metabolites on the regulation of acetyl-CoA carboxylase, and state the tissues in which fatty acid biosynthesis takes place. 10. State the roles of cytidine triphosphate and phosphatidic acid in the biosynthesis of phosphoglycerides. 11. Describe the functions of important phospholipids such as plasmalogens and platelet activating factor. 12. Describe the roles of the phospholipases A1, A2, C and D. 13. Describe the general principles of glycosphingolipid synthesis and degradation and the general features for pathogenesis of sphingolipidosis and their clinical presentation. 14. Name the most important sites of endogenous cholesterol synthesis and the most important intermediates of the biosynthetic pathway.

377

20.16

Biochemistry and Genetics

15. Know what an “isoprenoid” is. 16. Name the structural differences between cholesterol and the bile salts and describe the entero-hepatic circulation of bile salts and feedback inhibition of bile salt synthesis. 17. Identify conditions that predispose to the formation of gallstones. 18. Know the approximate lipid and protein content of the various lipoproteins. 19. Describe the metabolism of chylomicrons, VLDL, LDL and HDL. 20. Describe the mechanism of reverse cholesterol transport by HDL. 21. State the importance of LDL and scavenger receptors for the uptake of LDL by macrophages and parenchymatous cells. 22. Describe the regulatory effects of intracellular cholesterol on the LDL receptors and HMG-CoA reductase. 23. Outline the empiric relationship between lifestyle, lipoprotein levels and arteriosclerosis. 24. Describe the pathogenesis and clinical expressions of the major types hyperlipoproteinemia, including familial hypercholesterolemia. 25. Identify the mechanisms for the cholesterol and triglyceride lowering effects of cholestyramine, statins and fibrates and the effects of probucol as an antioxidant.

378

Part V.

Semester two, Mini II

21. Nutritional Management of Disease 21.1. Obesity Definition: Body weight > 20 % above ideal body weight. The body mass index (BMI) is used for estimating obesity: BMI = Weight (kg) / Height2 (m2 ) BMI > 27 indicates obesity. Weight gain occurs whenever energy input exceeds energy expenditure. Adipose tissue hyperplasia: Some obese patients including most of those with severe (“morbid”) obesity, have too many fat cells. The sites of adipose tissue hyperplasia are either abdominal (male-type) or hip and thigh (female-type). Adipose tissue hyperplasia may be caused by hypersensitivity to growth factors during childhood and/or puberty. This type of obesity, once established, responds poorly to dietary management. On a diet that maintains “normal” weight, patients have metabolic patterns as in starvation. Adipose tissue hypertrophy: Most patients with milder degrees of obesity have an increased amount of fat per adipose cell although the number of cells is more or less normal. This type is usually caused by over-eating. Appetite control: Stretch receptors in the stomach trigger satiety, as does an elevated blood glucose level. In addition, when adipose cells are “filled” they release the hormone leptin. Leptin acts on the brain to inhibit appetite, and it increases the metabolic rate. Causes of obesity: Genetic, heritability: ≈ 70 % in the US Bad eating habits: too many snacks and alcohol Eating for psychological reasons: stress, depression... Weight gain with each pregnancy. Poor weaning practices: use of too much sugar (empty calories!) at an early age Misuse of milk formula: too thick Low metabolic rate.

381

21.2

Biochemistry and Genetics

Lack of exercise. Complications of obesity: • Type 2 diabetes • Gallstones • Coronary heart disease • Hypertension Osteoarthritis and respiratory diseases may be exacerbated by obesity.

21.2. Weight Reduction Weight reduction leads to: • Lower blood pressure • Lower blood lipids • Lower blood sugar • Less heart disease Energy is required for: basal metabolic rate (BMR): 100.5 kJ/kg body weight in men and 90.4 kJ/kg body weight in women Physical activity: Between 20 % of BMR (sedentary lifestyle) and 50 % of BMR (heavy work) Postprandial thermogenesis: 10 % of the basal metabolic rate Example Mr. Jones weighs 90 kg, is 1.6 m tall, and has a sedentary lifestyle. Body mass index BMI = 90 kg / (1.6 m)2 = 90 kg / 2.56 m2 = 35.1 kg/m2 Basal metabolic rate BMR = 90 kg × 100.5 kJ/kg = 9045 kJ Physical activity: 0.2 × 9045 kJ = 1809 kJ Thermogenesis: 0.1 × 9045 kJ = 904.5 kJ Total energy expenditure: 11 759 kJ per day

382

Types of diabetes mellitus

21.3.1

How much should Mr. Jones eat if he wants to lose 1 kg per week? The energy content of adipose tissue is about 32.7 kJ/g (it’s about 85 % triglyceride). He has to reduce his weekly energy by 1000 g × 32.7 kJ/g / 7 d = 4671 kJ/d below his energy expenditure: 11 759 kJ/d - 4671 kJ/d = 7088 kJ/d daily energy intake. How much fat, CHO and protein should he eat? Fat should be 30 % of total energy, CHO 60 %, and protein 10 %. Fat: 0.3 × 7088 kJ/d / 38.9 kJ/g = 54.6 g, about a third each saturated, mono-unsaturated and poly-unsaturated. CHO: 0.6 × 7088 kJ/d / 16.7 kJ/g = 254.7 g, preferably from complex carbohydrates. Protein: 0.1 × 7088 kJ/d / 16.7 kJ/g = 42.4 g. Protein should have a high biological value. BMR will decrease because of loss of lean body mass: he will need fewer calories. Weight will reach a plateau unless calories are further reduced or exercise increased. Type of exercise Low impact: F 3–4 times/week I Intense walking, swimming, running, aerobics T 20–30 min/day at least

21.3. Diabetes Mellitus 21.3.1. Types of diabetes mellitus Type 1 diabetes (juvenile-onset diabetes) • Autoimmune destruction of β-cells • Onset in childhood or adolescence • Lifetime incidence 1 in 300 • Severe disease, insulin-dependence Type 2 diabetes (maturity-onset diabetes) • Poor tissue response to insulin (insulin resistance) and/or reduced glucose-stimulated insulin release • Adult-onset • Lifetime incidence 4–7 % • Less severe than type 1

383

21.3.2

Biochemistry and Genetics

Gestational diabetes occurs in some pregnant women because placental hormones antagonize the insulin effects. It does not persist after birth, but many patients develop type 2 diabetes later in life. Heritability: Moderate in type 1, high in type 2. Obesity: Most type 2 diabetics are obese. Most normal (non-diabetic) obese people have increased insulin levels with decreased tissue responsiveness. Signs and symptoms: • Hyperglycemia and glucosuria, from increased glucose production in liver and reduced utilization in liver, muscle and adipose tissue. • Increased plasma free fatty acids, from fat breakdown in adipose tissue. • Increased ketone bodies. These are formed from fatty acids in the liver. • Diabetic coma, with severe hyperglycemia, dehydration, electrolyte imbalances and, in type 1, acidosis (“ketoacidosis”) • Late complications: – Microangiopathy – nephropathy – retinopathy – peripheral neuropathy – lens opacities Importance: • It is the 4th leading cause of death - by disease. • It increases coronary heart disease risk 2–4 times. • Leading cause of kidney disease. • Leading cause of blindness. • Causes 1/2 of all leg amputations.

384

Management of Diabetes

21.3.2

21.3.2. Management of Diabetes Number 1 goal in management is improved metabolic control. This slows down the onset and progression of diabetic complications. Metabolic control is monitored by: • Blood glucose • Glycosylated hemoglobin • Blood lipids • Blood pressure • Renal function Goals in dietary management • Generally, to assist diabetics to make the necessary adjustments in their diets and exercise habits to ensure metabolic control. • Maintenance of normal blood glucose levels. • Attaintment of optimum serum lipid levels to reduce cardiovascular disease risk. • Provision of adequate calories to maintain or attain a healthy, realistic body weight. Adequate calories are needed to sustain physiological needs in pregnancy & lactation and to ensure optimal growth in children. • To prevent or delay long-term complications of the diabetic process; vascular angiopathies, gangrene, kidney disease, neuropathies, and blindness. • Education of patient and relatives. Overall dietary recommendations for diabetics Protein: 10–20 % of total energy intake Fat: 10 % from saturated, 15 % from monounsat. and 10 % from polyunsat CHO: 40–45 % of total energy intake If there is evidence of kidney damage (proteinuria), protein should be decreased to 10 % of energy intake., or 0.6–0.8 g/kg body weight. Levels of fat intake in diabetics: Type A: (Normal lipid levels) 10 % sat., 10 % mono., 10 % poly., 30 % of total energy intake Type B (Obese diabetic) 20 % of total energy intake from fat

385

21.3.2

Biochemistry and Genetics

Type C: (Elevated LDL levels) Fat < 30 % of total energy intake, Step II diet N.C.E.P., Saturated fat < 70 % 200 mg/d Type D: (Elevated triglycerides and VLDL) < 10 % saturated, 20 % monounsaturated, < 10 % polyunsaturated 20 % protein, 35–40 % CHO Including promotion of physical exercise and weight loss. Use of upomega-3 fats is strongly recommended: salmon, sardine, mackerel. Other aspects: Carbohydrates are still controversial. Meals with a low glycemic index are preferred. The glycemic index is the relative effect of a meal on the blood glucose level, compared with a standard food (glucose, or bread) of the same carbohydrate content. Sucrose shows a similar glycemic response to bread and potatoes: • Use with caution as part of the meal plan — about 5 % of the total energy intake. • Obese diabetics should try to abstain. • Fructose produces a smaller rise in plasma glucose levels than sucrose, but raises VLDL and LDL. Fruits and milk have a lower glycemic index than most starches. Legumes (peas and beans) have the lowest glycemic index and should be encouraged. Sodium: Assess on individual basis. • Diabetics with mild to moderate hypertension: 2400–3000 mg/d Na. • If there is kidney damage, 2000 mg/d or less: ‘No salt added’. Fiber: Certain types delay glucose absorption from small intestine, e.g. legumes. Beneficial effects on serum lipids. Exercise: beneficial. • Improves the body’s ability to use glucose. • Improves insulin responsiveness. • Lowers cholesterol. • Lowers blood pressure. • Reduces body fat. • Improves the circulation. • Reduces stress and gives a sense of well-being. Treatment for the obese non-insulin-dependent diabetic:

386

Coronary Heart Disease (CHD)

21.4

• Weight loss increases the number and responsiveness of insulin receptors. • Reduce total dietary fat. • Meal spacing at 4-5 h intervals • Realistic weight loss program: 2100 kJ/d or less. • Moderate regular exercise Treatment strategy for the type 1 diabetic: • Consistency in the quantity of food intake to match the amount of insulin. • Consistency in the timing of meals to match the onset and duration of insulin action. • Snacks are important, particularly at peak action of insulin (to prevent hypoglycemia) & before and after exercise. • Frequent self-monitoring of blood glucose is necessary if the patient is on intensive insulin therapy: 3–4 doses per day. • Alcohol should never be taken on an empty stomach. It impairs gluconeogenesis and can cause hypoglycemia. Gestational diabetes Diabetes in pregnancy is associated with fetal abnormality and stillbirths. Frequent monitoring of blood glucose is important. Carbohydrates calories must be adequate to prevent fat breakdown leading to ketosis. Reducing diets are not recommended during pregnancy. Recommended: 126–147 kJ per kg body weight per day with frequent snacks, particularly at bedtime.

21.4. Coronary Heart Disease (CHD) Pathology: CHD is caused by atheromatous plaques (atherosclerotic lesions) in one or more branches of the coronary artery. The plaque forms in the intima of the artery, typically with an inner core of cholesterol esters surrounded by fibrosis. It narrows the lumen of the artery and impairs blood flow. Result: exertional angina pectoris, with chest pain in response to physical activity (increased oxygen demand of the myocardium!). Or acute myocardial infarction, usually after the formation of a thrombus (intravascular blood clot) on the surface of the lesion. Atherosclerosis is also responsible for other ailments, including gangrene and some strokes. Prevalence of CHD: Myocardial infarction accounts for 25–40 % of all deaths in affluent countries but is rare in many third world countries. Atherosclerotic lesions take a long time

387

21.4.1

Biochemistry and Genetics

to develop, and therefore CHD is a disease of older people. Males are affected earlier than females. Development of lesions: The fatty streak is a benign, reversible precursor lesion which consists of lipid-laden macrophages and extracellular lipid (cholesterol ester) deposits in the intima. Fatty streaks are seen even in children. Most of them regress spontaneously, but some develop into atheromatous plaques, possibly by triggering the proliferation of neighboring smooth muscle cells. Major risk factors of CHD: • Smoking • Hypertension • Hypercholesterolemia • Diabetes mellitus Hypertension and diabetes are thought to facilitate the fibroproliferative response by “stressing” the vessel wall. A high LDL/HDL ratio leads to the accumulation of cholesterol esters. Lifestyle and diet affect the blood levels of LDL and HDL cholesterol, and thereby the risk of atherosclerosis and CHD.

21.4.1. Dietary Guidelines for Reducing Blood Cholesterol Eat less fat • Limit the use of fats in food preparation. Use as little butter, margarine and oil as possible. • Saute vegetables in 5 ml oil + 50 ml water instead of full fat. • Spoon off all fats after browning meats. • Trim off the visible fats on chicken and meat before cooking. • Keep salad dressings to minimum. Switch to low-fat dressings or “light” mayonnaise. • Use mint sauces (garlic, lemon, juice, herbs) rather than cream sauces. • Boil, steam, bake and poach, instead of frying. • Make wise food choices, for example: – Exchange whole-milk products for low-fat dairy products (low-fat cheese, e.g Mozzarella 10 % fat, Edema, Gouda or Camembert, milk and low-fat yogurt). – Use a low-fat spread

388

Dietary Guidelines for Reducing Blood Cholesterol

21.4.1

– Use unbuttered popcorn instead of peanuts. – Instead of fruit pies eat fresh fruit. – Eat low-fat buns instead of cakes and pastries. – Buy lean-cuts of meat and reduce serving sizes. – No sausages, bacon, minced meat, salami, luncheon meat, corned beef. SAMPLE MENU (25 % of total calories from fat): Breakfast: 1 fresh fruit, 2 slices whole wheat toast, 2 oz Mozzarella cheese, or: wholewheat cereal and fruit. Lunch: Tuna salad with lettuce, cucumber, whole wheat bread, 1 cup low-fat milk, fruit. Dinner: Grilled chicken, 1 teacup cooked brown rice boiled green beans, ground provisions. Stir-fry ratatouille, 1 cup low-fat milk, oat bran, date cookies or raisin buns. Use less saturated fat, i.e. 10 % of total energy intake. Fat type Appearance Sources Saturated Usually solid at room Butter, lard, palm oil, temperature veg. shortening, coconut oil, highly hydrogenated margarine, meat, poultry, cheese, egg yolk, dairy products, cocoa, coconut Monounsaturated Liquid at room tem- Canola, olive, and peanut perature oils. Peanuts, cashews, peanut butter and avocado Polyunsaturated Liquid at room tem- Safflower, sunflower, corn, perature soya bean, and cottonseed oils. Some margarines e.g. Becel, Fleishman, hazelnuts, almonds, mayonnaise made with veg. oil The trans-unsaturated fatty acids in hydrogenated vegetable fats (margarine, peanut butter) are physically, metabolically, and nutritionally similar to saturated fatty acids. It’s not the number of double bonds that counts, but the number of cis-double bonds!

389

21.5

Biochemistry and Genetics

Avoid (or reduce) high-cholesterol foods Dietary cholesterol should not exceed 300 mg/d. Plants do not contain cholesterol, therefore vegan diets are cholesterol-free. Food source Cholesterol (mg / 100g) Egg yolk 280 mg/yolk Organ meats (liver, kidney) > 350 Shrimp 159 Sardines 139 Crab, mackerel 103 Lobster 92 Meats: lamb, pork, beef, veal, poultry With skin, cod, game 76–94 Clams, oysters, scallop, tuna, halibut, trout 53–65 Salmon 41 Dairy cream 129 2 % fat milk 8 Whole milk 14 Ice cream 16 % butter fat 74 Cheese 96 Butter 620 Lard 91 Mayonnaise 160 Amount of cholesterol found in lb loaf: Commercial cakes = 110 mg Cheese cake 9” diameter = 2053 mg Increase the intake of dietary fiber, especially of “soluble fiber” from peas and beans. Use also fruit and oat bran. Whole grain cereals - whole wheat bread weetabix e.g. oatmeal, shredded wheat, alpen and vegetables are high in insoluble fibres which are particularly helpful for the regulation of bowels. Other measures to reduce blood cholesterol: • Quit smoking • Take regular exercise, at least 4 times/week, intensity will depend on tolerance levels • Lose weight, if you are overweight • Control blood pressure • Cut down on alcohol

390

Prevention of Coronary Heart Disease (CHD)

21.5

21.5. Prevention of Coronary Heart Disease (CHD) More than 500 000 Americans die from CHD annually. 1 250 000 Americans suffer from heart attacks each year. Costs range from US$ 50–100 billion annually, and nutritional prophylaxis and therapy is a cost-effective intervention. Risk factors for CHD: • High LDL cholesterol • Low HDL cholesterol • High plasma triglycerides • Cigarette smoking • Hypertension • Obesity • Lack of exercise • Family history • Diabetes • Race • Age • Gender

LDL cholesterol level <129 mg/dL Normal 130–159 mg/dL Borderline >160 mg/dL High risk Actual versus recommended U.S. dietary intake . Nutrient Present intake Population goals Fat 36–38 % total energy < 30 % total energy Saturated 13–14 % < 10 % Monounsaturated 15 % 15 % Polyunsaturated 10 % 10 % Cholesterol 230–260 mg < 300 mg

391

21.5

Biochemistry and Genetics

N.C.E.P National cholesterol education program . # of risk factors medical nutrition defined LDL goals therapy 2 or less Step I diet < 160 mg/dL 2 or more Step II diet < 130 mg/dL Established CHD Step II plus drugs < 100 mg/dL LDL cholesterol lev- Step II plus drugs Assess in 4 weeks, reels > 220 mg/dL assess in 12 weeks Continue to monitor, to ensure that aerobic exercise is maintained at least for 30 min 3–4 times per week. • Smoking should be discontinued • Weight loss should be actively encouraged • Maintain motivation and teach skills to manage the disease Nutrient Fat Protein CHO Saturated Fat Polyunsaturated Monounsaturated Cholesterol

Step I diet < 30 % of energy 15 % 55 % 10 % 10 % 15 % <300 mg/d

Step II diet Same Same Same <7 % Same Same <200 mg/d

Continue to monitor to prevent relapse into old habits. Reduced mortality from CHD has been recorded on vegetarian diets, more exercise and less alcohol. Monounsaturated fatty acids in Canola, olive and some brands of sunflower and safflower oil tend to increase HDL cholesterol. Heart disease is less prevalent in the Mediterranean region where olive oil is popular. Trans-unsaturated fatty acids in margarines, shortenings and peanut butter behave like saturated fatty acids and increase LDL while decreasing HDL. Substitution of polyunsaturated fats for saturated fats tends to decrease LDL, but has no effect on HDL. Excess polyunsaturates may pose a cancer risk and impair immunological function. Populations eating a lot of soy products have a low prevalence of CHD. Isoflavins (diadzein, genistein) are active ingredients and tend to lower LDL cholesterol. Vitamin C may increase HDL, lower total cholesterol and protect against LDL oxidation. Carotenes may reduce the risk of heart attacks: eat more carrots, broccoli, spinach etc. Like vitamins C and E, the carotenes are antioxidants.

392

Hypertension

21.6

Flavonoids are polyphenolics found in fruits, vegetables and red wine. They inhibit LDL oxidation and reduce thrombotic tendency. Supplements are not recommended.

21.6. Hypertension Importance: Hypertension is the chronic elevation of the arterial pressure. Normal: systolic pressure 120 mm Hg, diastolic pressure 80 mm Hg. Hypertension is common, usually of unknown origin (‘essential hypertension’) and with a strong genetic background. It causes microvascular changes, atherosclerosis and strokes. Blood pressure regulation: Blood pressure is determined by cardiac output and peripheral vascular resistance. Any increase in the blood volume will cause hypertension by increasing either cardiac output or peripheral vascular resistance or both. • Short-term regulation depends on the autonomic nervous system which adjusts cardiac output and peripheral vascular resistance. The sympathetic system accelerates the heart and causes vasoconstriction in many parts of the body, and the parasympathetic system slows down the heart and causes vasodilation. The autonomic nervous system is controlled by brainstem centers. Baroreceptors (pressure receptors) in the walls of aorta and carotid arteries provide specific input to these centers. • Long-term regulation adjusts the blood volume, and it depends on the kidneys. Important: the plasma contains fixed concentrations of electrolytes, mostly sodium and chloride, and these are in equilibrium with the interstitial fluid. Therefore any gain or loss of extracellular fluid volume (blood plasma + interstitial fluid) is possible only by adding or removing sodium and chloride (‘salt’). The normal kidney responds to increased blood pressure by excreting more water and salt (pressure diuresis). Also, when the blood pressure in the afferent arterioles of the kidney drops, the protease renin is released which generates angiotensin. Angiotensin is a hormone-like substance which increases the blood pressure by constricting arterioles throughout the body, thereby increasing the peripheral vascular resistance in the short term (within minutes). More importantly, it reduces renal blood flow, thereby reducing water and salt excretion directly, and it induces the release of aldosterone from the adrenal cortex. This hormone causes salt retention. Abnormalities in hypertension: Patients with essential hypertension have normal cardiac output but increased peripheral vascular resistance. They have a reduced renal blood flow because of high renal vascular resistance. Their kidneys can excrete a normal amount of salt and water, but only if the blood pressure is abnormally high. If their blood pressure is artificially lowered (for example by removing blood), salt and water excretion are reduced until the blood pressure has again reached an abnormally high level.

393

21.6

Biochemistry and Genetics

Essential hypertension has been linked to increased production of ouabain and digoxin in the adrenal gland. Both are glycosylated steroids involved in blood pressure regulation. Digoxin from plant sources has long been used to treat congestive heart failure in elderly patients (increases blood pressure and cardiac output, reduces heart rate). Dietary Management of Hypertension 60 million people in the US are hypertensive, 38 % Afro-American and 29 % White American. Essential Hypertension is classified according to the diastolic pressure: Mild 90–140 mm Hg Moderate 105–115 mm Hg Severe > 115 mm Hg Consequences of hypertension • Heart failure • Strokes • Heart attacks • Renal failure • Aortic aneurysm Risk factors for hypertension • Sedentary lifestyle • High-sodium diet • High-fat, low fiber diet • Obesity • Cigarette smoking • Excessive alcohol intake • Stress • Heredity

394

Hypertension

21.6

Manipulations which help to lower blood pressure • Low sodium intake • High potassium intake • High calcium intake • Increase in fiber • Increased unsaturated fat • Exercise • Relaxation • Reduced alcohol intake • Weight reduction

Sources of sodium 75 % of dietary sodium in the US comes from processed foods: cakes, biscuits, pretzels, pickled food, ketchup... Also some antacids contain sodium. Compare the sodium content of 3 oz fresh cooked pork: 59 mg Na+ 3 oz cooked ham: 114 mg Na+ Amount 1 teaspoon 1 oz 1 oz 1 cup 1/2 cup 1 oz 1 1 teaspoon 1 cup 1 level teaspoon 1 teaspoon 1 teaspoon 1 level teaspoon

Food Worcester sauce cheddar parmesan cheese chicken noodle soup canned tomato juice processed american cheese large olive soy sauce milk m.s.g baking powder bicarbonate of soda salt

Na+ (mg) 60 176 528 1100 440 400 130 330 120 500 370 1000 2000

395

21.7

Biochemistry and Genetics

General categories of sodium-controlled diets 250 mg/d Very severe, rarely used <1000 mg/d Severe 2400–4500mg/d Moderate 2000–3000mg/d Most commonly used Lower sodium intake leads to decreased blood pressure in sodium-sensitive people (10–20 % of the population), and reduced extracellular fluid volume.

21.7. The Liver Functional anatomy The liver receives a two-fold blood supply: the portal vein supplies blood that has passed through the capillaries of the splanchnic organs already; and the hepatic artery supplies fresh arterial blood. Water-soluble nutrients are absorbed from the small intestine into the blood and carried to the liver before they reach other parts of the body. Only lipids bypass the liver: they are transported in chylomicrons through the thoracic duct. The functional unit of the liver is the liver lobule, a cylindrical structure about 1 mm across and several mm long which is arranged around a central vein. Blood enters the lobule from the periphery and capillaries called sinusoids. The sinusoids have a fenestrated endothelium which facilitates the access of nutrients and other blood constituents to the hepatocytes. Functions The liver maintains a constant internal environment in the face of a fluctuating nutrient supply. It also serves many specialized functions: Regulation of blood glucose: The liver consumes glucose after a meal, converting the excess to glycogen and fat, and it produces glucose during fasting. Metabolism of non-nutritive food components (including drugs) to water-soluble products that can be excreted in bile or urine, alcohol is metabolized to acetic acid or oxidized to CO2 and H2 O. Storage of nutrients: Vitamin A, vitamin B12, iron. Conversion of toxic ammonia (NH3) to harmless urea. Production of bile Bile acids are important for fat absorption, and some waste products are excreted in the bile. Synthesis of plasma proteins (exception: immunoglobulins)

396

Disease of the Biliary System

21.7.2

Garbage removal: Worn-out plasma proteins and antigen-antibody complexes are removed by the liver, either by hepatocytes or by macrophages (Kupffer cells) in the walls of the sinusoids. Kupffer cells can also eat up bacteria that may enter the blood from the GI-tract.

21.7.1. Disease of the Biliary System The liver produces about 600–800 ml of bile daily. An amount of 40–70 ml is stored in the gallbladder. The bile contains bile salts, cholesterol, phospholipids, and bile pigments (glucuronic acid conjugates of bilirubin and other breakdown products of heme). Bladder bile is more concentrated than hepatic bile because electrolytes and water are absorbed from the bile through the gallbladder epithelium, thus increasing the concentrations of the other components. Fat in the duodenum stimulates the release of cholecystokinin, a hormone which induces contraction of gallbladder and subsequent release of bile into the common bile duct and into the duodenum. Most gallstones consist of cholesterol. They are associated with obesity. Obese patients with gallstones have too much biliary cholesterol. A high-fat diet may predispose to gallstone formation. Cholecystitis is inflammation of the gallbladder, caused by bacterial infection or gallstones. Some bacteria produce enzymes which deconjugate bilirubin. Unconjugated bilirubin is insoluble and forms pigment stones. Acute cholecystitis presents with pain, fever, vomiting and malaise. The diet should be fat-free and high in calories until the acute phase has subsided. A patient can become dehydrated and weak if these immediate steps are not taken. In chronic cholecystitis, a moderate fat-free diet should be given, pending surgery. Too much fat will cause the gallbladder to contract too frequently and cause the stones to shift. This can induce severe pain (colic). Cholestasis is interruption of the bile flow. It can be caused by hepatic disease (intrahepatic cholestasis) or obstruction of the common bile duct (posthepatic cholestasis. With fat intolerance, fatty stools and malabsorption of fat-soluble vitamins (vitamins A, D, E and K). Vitamin K deficiency appears early in acute cholestasis because this vitamin is not stored in the body, resulting in impaired blood clotting. Chronic cholestasis can lead to liver cirrhosis.

397

21.7.2

Biochemistry and Genetics

21.7.2. Parenchymal Liver Disease Fatty liver is a reversible lesion that appears whenever the liver produces more fat than it can export as VLDL. This happens in many liver diseases. Alcoholics get fatty liver because alcohol inhibits fatty acid oxidation, thereby diverting fatty acids into fat synthesis. People with protein-calorie malnutrition get fatty liver because their liver receives a lot of fatty acids from adipose tissue and some of them become esterified to fat, and because the synthesis of VLDL apo-proteins is impaired by amino acid deficiency. Liver cirrhosis is the outcome of many liver diseases. Most cases result from chronic alcoholism and/or chronic active hepatitis. Cirrhosis may also result from liver toxins such as carbon tetrachloride or drugs, and from severe acute viral infections (hepatitis, yellow fever). In the cirrhotic liver, much of the normal tissue has been replaced by fibrous connective tissue. The cirrhotic liver may appear as a firm fibrous mass with orange colored nodules projecting from its surface. Blood flow is impaired, causing portal hypertension. This results in development of a collateral circulation through retroperitoneal veins, periumbilical veins, lower esophageal veins, and rectal veins. These veins dilate. Some patients develop dangerous (often fatal) esophageal bleeding. Blood clotting is impaired formation of clotting factors. Signs and symptoms include abdominal edema (ascites), jaundice, vitamin deficiencies, encephalopathy (impaired intellectual function with confusion and stupor), nausea, vomiting, anorexia, and epigastric pain. Biochemical features include • Raised serum bilirubin • Low serum albumin • Raised serum alkaline phosphatase • Raised serum transaminases • Prolonged prothrombin time • Raised serum ammonia Treatment is symptomatic. In the absence of impending coma and as long as blood ammonia is normal, a protein-saving diet (enough energy from CHO and fat that protein-catabolism is not required, enough protein of high biological value to cover anabolic needs) is recommended in order to correct severe undernutrition, regenerate functional liver tissue and replenish plasma proteins. However, if signs of encephalopathy or hepatic coma appear, the protein is adjusted to individual tolerance. Recommended sodium: 500 −-1000 mg/d Na+ . Vitamins must be supplied according to individual need and deficiency. Moderate fat is used. Alcohol is strictly forbidden.

398

The Kidney

21.8

Hepatic coma is seen in patients with advanced liver cirrhosis, and also in patients with severe acute liver diseases (toxins, viral infections). It results from the inability of the severely diseased liver to maintain a normal internal environment. Ammonia accumulation is especially dangerous. Gastrointestinal bleeding can precipitate hepatic coma because it supplies intestinal bacteria with extra protein which is fermented to ammonia. Hepatic coma develops when ammonia-laden blood cannot pass through the cirrhotic liver and is rerouted through the collateral circulation. It re-enters the systemic blood flow, still carrying its ammonia load, and produces ammonia intoxication. Clinical manifestations of hepatic coma include apathy, confusion, inappropriate behavior, drowsiness, ‘absent stare’, slurred speech, and tumor in the outstretched hand. The breath may have fecal odor. Treatment is aimed at the prevention of bleeding and reduction of ammonia formation. Dietary Management Protein must be withdrawn from the diet completely and energy requirements met by CHO and fat, initially parenterally through a peripheral vein. Example: 1–2 l 10 % dextrose in 24 h. Parentrovite I and II, No Intralipid. Simultaneously a nasogastric feed may also be given to provide energy.

21.8. The Kidney Functions The kidneys play a key role in homeostasis (maintenance of a constant internal environment) and in the excretion of waste products: • Excess water and electrolytes are excreted. Thereby the kidneys controls extracellular fluid volume (plasma + interstitial fluid) and the correct ionic composition of the plasma. • Protons are excreted or retained to maintain the blood pH at the normal value of 7.4. • Nitrogenous wastes are excreted: urea from amino acids, uric acid from purines, creatinine from creatine. Urea excretion is directly proportional to the dietary protein intake. • Many drugs are excreted by the kidneys. • The kidney is also an endocrine organ. It produces: Calcitriol the active form of vitamin D Renin a protease that makes angiotensin Erythropoietin a growth factor that stimulates RBC formation in the bone marrow

399

21.8.1

Biochemistry and Genetics

Functional anatomy The kidneys weigh only 300 g (0.4 % of the body weight) but receive 1.2 l blood per minute (25 % of the cardiac output). The functional unit is the nephron. The nephron starts with a small capillary bundle called glomerulus which is embedded in a cup-like structure called Bowman’s capsule. Water, electrolytes and small molecules are filtered into the space between glomerulus and Bowman’s capsule, and blood cells and plasma proteins are retained in the blood. About 150–200 l of fluid are formed this way every day. This primary filtrate is passed through a long, convoluted tubular system where useful substances (glucose, amino acids) and most of the water and electrolytes are retrieved by active transport across the tubular epithelium. Also protons are transported across the tubular epithelium, and under most Conditions the urine is neutral or mildly acidic (pH 5.0–7.0).

21.8.1. Glomerulonephritis and Nephrotic Syndrome Acute glomerulonephritis is an acute immunological response, usually after a streptococcal sore throat. There is acute inflammation of the glomeruli with congestion of renal blood flow and reduced glomerular filtration rate. Typical features: • Reduced urinary volume • Proteinuria, presence of blood cells in the urine, casts of protein precipitated in the tubular system • Edema of face and hands in the morning and ankles in the evening • Raised blood pressure, headache, malaise Treatment • Drugs for hypertension and diuresis • Fluid intake must be carefully monitored • 1000–1500 mg sodium/day • Protein restriction to 40 g/d if blood urea nitrogen (BUN) is high Sample meal plan: Milk 0.25 l/d Breakfast 1 egg, Toast and tea, Fruit juice Lunch 50 g meat or fish salt free, small portion of vegetables, Rice cooked without salt Dinner Salt-free meat or chicken (30 g), Bread and low-salt margarine

400

End-Stage Renal Disease

21.8.2

Nephrotic syndrome is characterized by proteinuria, hypoalbuminemia, and peripheral edema. The glomerular basement membrane is damaged, and plasma proteins are lost in the urine. Some patients lose 20 g of protein per day, and their serum albumin level drops to 1.5 g/dL. It can occur as a complication of malarial infection, following certain types of acute glomerular nephritis, in diabetes, autoimmune diseases (systemic lupus erythematosus), and renal vein thrombosis. Nephrotic syndrome can progress to renal failure, even if it is under control. The loss of fluid from the vascular space into the interstitium reduces blood volume and blood pressure. As a homeostatic response, the renin-angiotensin system is activated, and the release of aldosterone causes the retention of sodium and water. Dietary management plays an important role, along with diuretics and anti-inflammatory steroids. Protein: 1 g protein/kg ideal body weight plus 1 g protein for every gram lost in the urine. Energy: 840 J for every gram of nitrogen lost can achieve satisfactory nitrogen balance. Sodium: 1800–2300 mg/d. Many patients have hyperlipidemia. This should be treated by replacing animal fat with polyunsaturated fat.

21.8.2. End-Stage Renal Disease Renal failure can be caused by pyelonephritis, glomerulonephritis, hypertension, polycystic kidney disease, obstruction of the urinary tract, and diabetes mellitus. Major problems: • Uremia, with accumulation of nitrogenous waste products and with general and especially CNS toxicity • Water retention, with increased blood volume and hypertension • Phosphate, magnesium, and potassium are retained. But calcium may be low because of impaired intestinal absorption (lack of calcitriol!). • The bones become demineralized (renal osteodystrophy) because of chronic acidosis and low calcitriol. • Anemia develops, because erythropoietin is missing. • Drugs that are normally excreted by the kidneys are poorly tolerated by patients with renal failure. The dietary management of renal failure is based on the restriction of everything that is normally excreted by the kidneys: potassium, phosphate, magnesium, protein (urea formation!). Specific recommendations:

401

21.9

Biochemistry and Genetics

Protein: Protein should be restricted to prevent the accumulation of nitrogenous wastes. However, uremic patients have increased protein breakdown as a result of uremic toxicity, chronic acidosis, and high levels of stress hormones (glucocorticoids!), therefore they are prone to develop a negative nitrogen balance. Current recommendation for conservative management is 0.6 g protein/kg body weight. This may be supplemented with small amounts of essential amino acids or their alpha-keto acids. 70 % of protein should be of high biological value. It should be spaced out evenly over the day to allow better utilization. Milk should be included to provide a source of calcium. Nuts and beans are not recommended because of their high potassium and phosphate content. Energy: 170 J/kg body weight. Carbohydrate should never be in short supply, to prevent excessive breakdown of body protein. Sodium: 1500–2000 mg/d. No added salt. Potassium: 1500–3000 mg/d, about 600 mg should come from vegetables and fruits low in potassium. Potassium-containing salt substitutes must be avoided. Phosphorus: Restriction of protein will automatically reduce phosphorus. Use bran with caution because of its high phosphate content. Many uremic patients have a type IV hyperlipoproteinemia. This should be treated by replacing animal fat with polyunsaturated fat.

21.9. Nutritional Therapy In Surgery And Injury The trauma associated with surgery always increase the need for nutrients and the circumstances usually restrict food intake temporarily. Following surgery there is a catabolic phase, and certain hormones are secreted. Vasopressin causes water retention. Aldosterone reduces renal blood flow. Glucocorticoids induce protein breakdown in many tissues, and gluconeogenesis in the liver. Thyroxine increases the metabolic rate. Epinephrine and norepinephrine induce fat breakdown in adipose tissue and glycogen breakdown in muscle and liver. The increased metabolism requires nutrients, and there may be extra requirements for the replacement of losses: hemorrhage, fistulae, serous exudates from burns. During fever, basal metabolic rate increases by 10 % for every degree centigrade rise in body temperature. Water: Adequate fluid is necessary to prevent dehydration. Large water losses may occur from vomiting, hemorrhage, fever, exudates, and diuresis. A total of 2000 ml water per day is needed in uncomplicated cases, but up to 7000 ml in complicated cases, such as drainage or sepsis.

402

Nutritional Therapy In Surgery And Injury

21.9

Protein: this is the most important nutritional concern in surgical patients. A negative nitrogen balance of up to 20 g/d may occur. Added protein losses may occur in hemorrhage or exudates. Protein synthesis is required for • Wound healing • Maintenance of plasma protein levels, prevents circulatory shock and edema • The synthesis of immunoglobulins and other proteins for body defenses The protein intake should be near 1.9 g/kg/d in uncomplicated cases to 2.5 g/kg/d in cases of severe stress. Energy: An adequate amount of carbohydrate is essential to ensure the use of protein for building tissue and to supply the energy required for increased metabolic demands; 170–210 J/kg/d. Vitamins: Vitamin C is necessary for collagen synthesis during wound healing; B vitamins are coenzymes for enzymes in energy metabolism; and vitamin K is needed for blood clotting.

403

22. Amino Acid Metabolism 22.1. Protein metabolism About 300 g of protein are synthesized each day in our body, this is balanced by proteolysis of an equal amount of protein. This turnover is required in part by replacement of proteins within cells, but also to form new cells in organs with rapid cell replacement (e.g. blood, intestinal epithelium) and requires about 5 % of our basal metabolism. More protein synthesis is required in growth, pregnancy, lactation or to build muscle mass in training athletes. Amino acids are also required for the synthesis of metabolites like heme, hormones, nucleotides, coenzymes, melanin and biogenic amines. To ensure an adequate supply of amino acids it is recommended that adults eat about 0.6– 0.8 g kg−1 d−1 protein, for infants this rises to 2.2 g kg−1 d−1 . Protein intake above these recommended values does not offer any advantages with respect to strength or overall health, but is associated with several health risks (for a review see [Metges and Barth, 2000]). However, available data do not yet permit setting upper intake levels. Our body can not store free amino acids, any excess has to be catabolized. This generates 17 kJ/g metabolic energy. To assess nutrition with respect to protein we look at the balance between nitrogen • uptake (≈ 16 % of protein intake)

Figure 22.1.: Nitrogen balance in an average US-citizen. Amino acids taken up with daily food are largely catabolized, producing urea, glucose and ketone bodies. urea Body protein 250-300 g/d

Dietary protein

~ 100 g/d

NH 3

250-300 g/d

Amino acid pool

~ 100 g/d

Glucose Carbon chains

Ketone bodies CO2 + H2 O

Metabolites

405

22.1.1

Biochemistry and Genetics

• loss – urine – feces – milk (lactating ~ only) – “other losses” (≈ 5 mg kg−1 d−1 ) Then there are three possible situations: uptake > loss positive balance (growth, pregnancy) uptake = loss equilibrium uptake < loss negative balance, wasting of tissue (malnutrition, stress)

22.1.1. Biological value of proteins There are 20 common proteinogenic amino acids (plus selenocysteine and pyrrolysine, which we do not discuss in this chapter). Of these, most can be produced in our body, however, some can not and need to be taken up, these we call essential amino acids: Amino acid amount (g/d) Arginine children only Histidine unknown Isoleucine 1.30 Leucine 2.02 Lysine 1.50 Methionine 2.02 Phenylalanine 2.02 Threonine 0.91 Tryptophane 0.46 Valine 1.50 Arginine can be produced in our body (as we will see later), but the amount is insufficient to support rapid growth in children. Histidine is stored in muscle cells as carnosine, a short peptide (βAla-His) which serves as buffer substance. In case of His-deficiency breakdown of carnosine will prevent a negative nitrogen balance for a long time, therefore the daily need could not be established experimentally. Because we can not store free amino acids all of them need to be supplied in the required amounts at the time of protein synthesis. The protein in our food therefore needs to have approximately the same amino acid composition as the proteins in our body, we say: it should have a high biological value. There are several ways to express the biological value of a protein, the most important of these are:

406

Biological value of proteins

22.1.1

protein score for all amino acid determine contend/required ratio, take lowest value. NPU (net protein utilization): ratio of nitrogen retained to nitrogen supplied. Takes digestibility into account. Here are the values for some important food sources: Source protein score NPU Human milk 1.00 0.95 Beef steak 0.98 0.93 Egg 1.00 0.87 Cow milk 0.95 0.81 Corn 0.49 0.36 white rice 0.67 0.63 Wheat 0.47 0.30 In general, the closer a species is related to us evolutionary, the closer its protein composition will be to our own, and the higher the biological value of its proteins.

Mixing of food to achieve high biological value

Soy protein has the following composition: Amino acid content (g/100g) required (g/d) ratio Arginine 7.72 Histidine 2.33 Isoleucine 5.31 1.30 4.08 Leucine 7.98 2.02 3.95 Lysine 6.65 1.50 4.43 Methionine 1.40 2.02 0.69 Phenylalanine 5.08 2.02 2.51 Threonine 3.90 0.91 4.29 Tryptophane 1.53 0.46 3.33 Valine 5.34 1.50 3.56 The protein score is 0.69, with Met being the limiting amino acid. Rice on the other hand has the following composition:

407

22.2.1 Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophane Valine Thus the protein

Biochemistry and Genetics

content (g/100g) 7.90 2.70 4.10 8.90 4.00 3.20 5.50 3.90 0.31 6.50 score is 0.67, with

required (g/d) ratio 1.30 3.15 2.02 4.41 1.50 2.67 2.02 1.58 2.02 2.72 0.91 4.29 0.46 0.67 1.50 4.33 Trp being the limiting amino acid.

Neither soy nor rice, both important food sources, could serve human nutritional needs alone. A mixture of both on the other hand would serve beautifully, the Met being supplied by rice and the Trp by soy. Thus it is not necessary that each individual foodstuff has the ideal mixture of amino acids, but the overall mixture in a meal should have. Many indigenous people with limited access to animal protein use this principle, for example soy/rice in Asia and beans/corn in the Americas. Note however that since amino acids can not be stored the supply of essential amino acids can not be distributed over several meals.

22.2. Nitrogen metabolism 22.2.1. Nitrogen transfer If more amino acids are supplied with food than required for protein synthesis, or if the amino acids in food can not be used completely because of poor biological value, the remaining amino acids must be catabolized as they can not be stored in our body. Catabolism occurs in two steps: first the amino group is removed and converted to urea. Second the carbon skeleton is degraded. The latter step will be discussed in the next section. To remove the amino group from an amino acid our body has three possibilities (see fig. 22.2): transamination In this reversible reaction the amino group is transferred onto a keto-acid, usually α-ketoglutarate. The reaction products are the ketoacid corresponding to the amino acid and glutamate. Lys, Thr and Pro can not undergo transamination. Their metabolism will be discussed in the next section.

408

22.2.1

Nitrogen transfer

Figure 22.2.: Metabolism of amino acids by transaminases, oxidases and glutamate dehydrogenase. For details see text. α-ketoglutarate

not: K,T, P

COO COO H3N

+

-

COO

O C

+

CH

COO

CH2 CH2

R

COO

HO H3C

H3N

H2 C O Pi

N

H3C

+

H2O

NH4+

NAD(P)+

NAD(P)H + H+

CH2

-

+

CH2

H2 C O Pi

N

ping-pong-mechanism

-

CH2 CH2

-

COO

COO

-

Remember: NADPH is used for anabolic pathways (NADPH/NADP + > 1) NAD+ is used for catabolic pathways (NADH/NAD + < 1)

-

O2 H2O

H2O2 NH4+

Glu COO

+

HC N H3 R

CH2

O C Glutamate dehydrogenase

CH2 COO

+

Pyridoxamine phosphate

COO

CH CH2

-

CH

COO

HO

Pyridoxal phosphate (Vit B6)

H2N

H2N

R

Transaminase

-

CH

-

+

-

O C

O

COO

glutamate

ketoacid

-

O C d-amino acid oxidase

α-ketoglutarate

-

R

COO transaminase

H3N

+

-

CH R

no longer chiral !

409

22.2.2

Biochemistry and Genetics

Transamination is the archetypical example for a ping-pong reaction mechanism (see section 7.6 on page 161). Transaminases contain pyridoxalphosphate, the active form of vitamin B6 . The amino acid is bound, gets the keto-oxygen in exchange for its amino group and leaves. The enzyme now has pyridoxamine phosphate in its active center, after binding of α-ketoglutarate it will transfer the amino group onto this compound, in exchange for the keto-group. Release of Glu closes the reaction cycle. oxidation by amino acid oxidases is used to convert d-amino acids (for example from the murein sacculus of bacteria) into the corresponding l-isomer. The amino acid is oxidized to the corresponding α-ketoacid using molecular oxygen. Side products are ammonium and hydrogen peroxide. Hydrogen peroxide is a reactive oxygen species (ROS), which needs to be detoxified (see section 15.3 on page 253). The reaction is localized in the peroxisome. The ketoacid is no longer chiral, it can either undergo degradation, or it can be converted into the corresponding l-amino acid by transamination. glutamate dehydrogenase cleaves the amino group from Glu by reducing nicotinamide. The reaction is reversible and can be used both for the degradation and production of Glu. However, cleavage of Glu into α-ketoglutarate and ammonia is performed by reducing NAD+ to NADH + H+ while the production of Glu from α-ketoglutarate and ammonia is performed by oxidizing NADPH + H+ to NADP+ . The reason is that in the cell NAD+ > NADH + H+ (which favors reduction of the nucleotide) while NADP+ < NADPH + H+ , which favors its oxidation. Remember that this allows the independent regulation of catabolic and anabolic pathways!

22.2.2. Urea-cycle As we have seen in the previous subsection, amino acids are catabolized by removing their amino group, producing ammonium ions in the process. Ammonium ions however are quite toxic, they have to be removed from the body. Since ammonium has limited water solubility, only aquatic organisms can afford to excrete it directly via the gills. Land-living organisms have to convert it into a suitable form to remove it via the kidneys. Mammals use the highly water-soluble urea. Reptiles and birds use uric acid instead, this compound is almost water insoluble. Their “urine” is therefore a paste, saving weight and water. Production of urea occurs in the liver, it is a very energy intensive process (see fig. 22.3). For that reason some of the enzymes of this pathway are located in the mitochondria. Urea production occurs in several enzymatic steps: carbamoylphosphate synthetase I transfers the ammonium onto carbon dioxide, forming carbamoylphosphate. This reaction requires 2 molecules of ATP and occurs in the mitochondria. The ammonium comes mostly from glutamate dehydrogenase reactions in

410

22.2.2

Urea-cycle

Figure 22.3.: The urea cycle. For details see text. this nitrogen mostly from blood

Urea cycle

NH4+ + CO2 + 2 ATP + H2O Carbamoylphosphate synthetase I 2q35, 1:800,000, early and late onset form

+

2 ADP + Pi + 3 H+

Deficiency in general: hyperammonemia, lethargy coma, agitation, Reye-syndrome like encephalopathy

Liver mitochondria

O H2N

C

O

Pi Pi + H+

NH2 CO

+

NH

N H3 CH2 CH2 CH2

HC H3N

Urea

+

CH2 CH2

+

HC H3N

-

COO

Ornithine

Citrulline

COO

O C

CH2

Ornithine transcarbamoylase Xp21.1, recessive, female may be affected (X-inactivation, Barr-body) 1:100,000

NH2

progressive spastic quadriplegia, mental retardation 6q23

NH2

Arginase

+

C NH2 NH CH2

Argininosuccinate aciduria: 7cen-q11.2, treatment: Arg + Ornitine Argininosuccinate lyase

CH2

AMP + PPi + 2 H+ AMP + ATP +

2 ADP

-

N H2

COO

C N CH H CH NH -

COO

CH2 CH2

CH2

+

HC H3N Arginine

this nitrogen mostly from transamination in the liver

chromosome 9q34 1:100,000

Liver cytosol

N H2

COO

ATP + Aspartate Argininosuccinate synthetase Citrullinemia

Induction with Shope papilloma virus?

H2O

-

-

Fumarate

CH2

HC H3N COO

+

-

Argininosuccinate

Summary: NH4+ + CO2 + 4 ATP + 2 H2O + Aspartate = 4 ADP + 4 Pi + 5 H+ + Furmarate + Urea

411

22.2.2

Biochemistry and Genetics

extrahepatic tissues, especially muscle. In the form of ammonia it is freely membrane permeable (small gas molecule!). ornithine transcarbamoylase catalyzes the transfer of the carbamoyl-group onto ornithine, forming citrulline in the process. Ornithine is transported into, and citrulline out of the mitochondria by specialized transporters. argininosuccinate synthetase transfers aspartate onto the citrulline. Note that the red carbon in fig. 22.3 now carries two nitrogens, just as it will in urea. The reaction again requires energy in the form of ATP. However, the ATP is split into diphosphate (pyrophosphate, PPi ) and AMP. Since the PPi is immediately hydrolyzed by pyrophosphatases, its concentration in the cell is very low. This reduces the speed of the backward reaction (remember the principle of Le Chatelier!). We will see other examples of this expensive trick as we progress through metabolism. Mitochondrial ATP-synthase can not handle AMP as substrate, it has to be converted first to ADP by adenylate kinase: ATP + AMP * ) 2ADP. Thus the reaction actually consumes a total of 2 molecules of ATP. The nitrogen of the aspartate used comes mostly from transamination reactions in the liver. argininosuccinate lyase cleaves fumarate from the argininosuccinate, forming arginine (remember from above that humans can make their own arginine?). arginase cleaves the guanidino-group from arginine forming urea and ornithine. The ornithine can be re-used for the synthesis of another urea (hence urea-cycle). Note the difference between synthase and synthetase: the latter requires energy in the form of ATP. The total reaction of urea production is: + − − NH+ 4 + 4 ATP + 2 H2 O + Aspartate → 4 ADP + 4 Pi + 5 H + Fumarate + H2 N CO NH2 . Control of urea cycle • increased amino acid breakdown • increased liver [Glu] • production of N-acetyl glutamate • which allosterically activates carbamoyl phosphate synthetase Urea cycle failure results in hyperammonemia and encephalopathy. Especially in complete deficiency the prognosis for these patients is poor. There are two possible reasons for urea cycle failure: inherited Feeding difficulties, vomiting, ataxia, lethargy, Reye-syndrome like encephalopathies, coma, death. Induced aversion against protein-rich foods in mild cases.

412

22.2.2

Urea-cycle

Figure 22.4.: Nitrogen sources for the urea pathway. Most of the nitrogen on the aspartate comes from transamination reaction in the liver, while the ammonium comes from extrahepatic sources via blood. However, if either is not available in sufficient amounts to get rid of the other, conversion is possible. not K, T, P

α-ketoacid

amino acid transaminase

NH4+ + α-ketoglutarate

glutamate

glutamate

dehydrogenase

α-ketoglutarate aspartate transaminase

oxaloacetate

aspartate

furmarate

urea

liver cirrhosis Alcoholism, viral hepatitis. Ataxia, slurred speech, tremor (asterixis), mental derangements (Wernicke-Korsakoff-syndrome).

In either case mainstays of treatment are an alcohol free and protein reduced diet in which essential amino acids are replaced by their α-ketoacids. Thus the nitrogen is recycled rather than excreted. Two substances can aid in treatment:

• Benzoate + Gly → Hippurate

• Phenylacetate + Gln → Phenylacetyl-Gln

Hippurate and Phenylacetyl-Gln are excreted in urine.

For all enzymes involved in the urea cycle defect mutants have been reported in human patients:

413

22.3

Biochemistry and Genetics

Enzyme Carbamoylphosphate synthetase I Ornithine transcarbamoylase

location 2q35 Xp21.1

frequency 1:800 000 1:100 000

Argininosuccinate synthetase argininosuccinate lyase

9q34 7cen-q11.2

1:100 000 1:70 000

Arginase

6q23

1:1 000 000

remark early and late onset forms X-linked, but with Xinactivation in ♀ possible citrullinemia argininosuccinate aciduria, treatment with Arg + Ornithine progressive spastic quadriplegia

22.3. Catabolism of the carbon backbone After removal of the amino group the charbon backbone of the amino acids need to be converted into compounds that enter the pathways of glucose and/or fatty acid metabolism. Amino acids are divided into 3 groups depending on where their backbone enters general catabolic pathways: glucogenic amino acids feed into glycolysis and Krebs-cycle: Pyruvate Ala, Cys, Gly, Ser Oxaloacetate Asp, Asn α-ketoglutarate Arg, Glu, Gln, His, Pro Succinyl-CoA Met, Val ketogenic amino acids produce acetyl-CoA (Leu, Lys) glucogenic and ketogenic produce precursors of gluconeogenesis as well as ketone bodies (Ile, Phe, Tyr, Trp, Thr). Usually the ratio of these precursors is fixed, only Thr can be used to make either glucose or ketones. Amino acids are an important source of glucose and ketone bodies during starvation and physical exertion. In these cases body protein, in particular muscle protein, is broken down. The glucose is used to maintain a constant blood glucose concentration, which is required for the correct function of brain, erythrocytes and kidney cortex. The ketone bodies are used as fuel for muscles, especially the heart. For further discussion of this homeostatic mechanism, see chapter 31 on page 569.

414

Ala and Ser enter glycolysis

22.3.1

Medical application: The low carbohydrate, high protein (Atkins-) diet This diet was introduced to help overweight and obese patients with weight reduction. Given the increasing morbidity from metabolic syndrome in developed societies that is an important goal. Let us however consider the biochemical consequences of such a diet. Our body requires glucose as essential nutrient. Brain, erythrocytes and kidney cortex depend on a constant supply of glucose for their correct function, a relatively small drop in blood glucose concentration results in life-threatening hypoglycemic coma. In case of a person on a low carbohydrate diet the body has to maintain blood glucose level by gluconeogenesis from proteins. This is actually one of the mechanisms by which the Atkinsdiet is supposed to work: Gluconeogenesis is metabolically expensive, the energy spend on doing this is no longer available to make body fat. However, proteins contain both glucogenic and ketogenic amino acids, and since amino acids can not be stored both of them have to be metabolized at the same time. This leads to ketoacidosis, which in turn has a diuretic effect. The drop in blood pH together with dehydration and loss of minerals can result in a dangerous physiological situation, in extreme (and very rare) cases leading to coma and death. It is the diuretic effect of the Atkins-diet that results in the initial rapid weight loss (1-2 kg over a few days) of people on this diet, which is no doubt one explanation for the popularity of this diet. The difference between loss of water and loss of fat is not necessarily clear to all dieters. Every diet results not only in a loss of fat, but also of lean body mass in form of muscle protein, which can only be partly prevented by increased physical activity. In a Atkins-type diet this effect is more pronounced than in a balanced, calory-reduced diet. In addition, the lack of glucose puts the body into an “energy saving mode”, which makes weight loss more difficult (see chapter 31 for the mechanism). It is probably all these factors that conspire to make the drop-out rates in Atkins-type diets higher than in conventional ones (see Westmann et al., 2002 for a prospective study jointly done by members of the Atkins-institute and critics of this diet).

22.3.1. Ala and Ser enter glycolysis Alanine The metabolism of Ala is particularly simple, transamination converts it into pyruvate. Since transamination is reversible, Ala can also be produced from pyruvate. The enzyme responsible, alanine:glutamate transaminase, also known as glutamate:pyruvate transaminase (GPT), is located in liver cytosol. In case of necrotic liver damage the enzyme is released into the blood stream, where it can be detected by appropriate laboratory tests with high sensitivity at low costs (see section 10.4 on page 208 for details). Detection is by

415

22.3.1

Biochemistry and Genetics

Figure 22.5.: Alanine and serine are produced from and converted into metabolites of glycolysis. For details see text. Ser-biosynthesis O

O

NAD+

C

3-Phosphoglycerate

NADH + H+

HC OH O

O

C

O

H3N

C O P O H2 O

O

CH

O

3P-Serine

Pi

C +

O

NADH + H+ NAD=

C

αKG

C OH H2

C OH H2 O

Serine

O

O C

HC OH

O

O

CH C OH H2

D-Glycerate C

C

H3N

3-Hydroxypyruvate

O

O

O

O

O

O

C O P O H2 O H2O

3P-hydroxypyruvate

O

Phosphoenolpyruvate

+

ATP

HC O P O HO CH2

O

αKG

C

O C

ADP

O

Glu

C

C O P O H2 O

2-Phosphoglycerate

O

Glu

Gluconeogenesis

C O P O O

CH2

αKG

Glu

O

O

O C +

H3N

O

Alanine

alanine glutamate transaminase

this enzyme is of diagnostic significance (GPT)

H3N

Pyruvate

Gly

Cys

+

O C

CH2

O C CH3

(Thr) Trp

H 2O

H 2O NH4+

O

O

C

O C

CH CH3

416

Serine dehydratase

C H2N

+

C

CH3

22.3.2

Glu, Gln, Asp and Asn enter the Krebs-cycle

Figure 22.6.: Transamination of Glu and Asp produces α-ketoglutarate and oxaloacetate respectively, which are intermediates of the Krebs-cycle. Gln and Asn are produced from Glu and Asp by amidation of the terminal carboxylic acid group in an energy-requiring reaction. Also shown is where other amino acids enter the Krebs-cycle. Phe

Tyr

Trp

Lys

this enzyme is of clinical significance (AST) αKG

Aspartate O H 3N

+

Asparagine synthetase

C

S CoA

Glutamine O

C

H3N

O

O

C

+

C

C

NH2

ADP Pi Glutamine

Glutaminase

NH4+ ATP

NH4+ L-Malate

O

C

O

O H3N

+

O C

C

Furmarate

O O 2-Oxoglutarate

Tyr

Succinate

Succinyl-CoA

Met

Ile Thr

Val

C

aa

Glutamate: oxoglutarate aminotransferase

Asparagine

O

CH CH2

CH2

Phe

C

synthetase

CH2

CH2

CH2 O

NH2

O

H2O

iso-Citrate

Oxaloacetate

O

CH

+

C

O

H2O O

O

CH CH2

NH4+ Asparaginase

C

H 3N

cis-Aconitate

C

O

AMP PPi

+

CH2

aspartate glutamate transaminase

CH

NH4+ ATP

H 3N

O O

CH2 O

H 3C C

Citrate

Gl

O

C

Leu

O

Ile

ka

O

O

Glutamate Arg

His

Pro

this enzyme is of clinical significance (GOT)

coupling GPT and glutamate dehydrogenase in an optical test (see section 7.9 on page 169 for how this works).

One of the most frequent causes for liver damage is alcohol consumption, a single glass of alcoholic beverage kills enough liver cells that the enzymes released can be detected for 2–3 days. This is of forensic importance: Driving under the influence is usually punished by withdrawal of the drivers licence, which is returned only after the culprit has demonstrated that (s)he can live over extended periods without alcohol.

Serine Serine is produced from the glycolytic intermediate 3-phosphoglycerate by reduction, transamination and dephosphorylation (see fig. 22.5). The latter step is irreversible, to catabolize Ser into 3-PG a different pathway is used, which uses ATP. Serine may also be converted to pyruvate via serine dehydratase.

417

22.3.3

Biochemistry and Genetics

22.3.2. Glu, Gln, Asp and Asn enter the Krebs-cycle Transamination of Glu and Asp produces α-ketoglutarate and oxaloacetate respectively, which are intermediates of the Krebs-cycle. Like GPT the enzymes involved, glutamate:oxoglutarate and aspartate:glutamate aminotransferase respectively, are used as diagnostic marker for liver damage (GOT and AGT, also abbreviated AST). Gln and Asn are produced from Glu and Asp by amidation of the terminal carboxylic acid group. This reaction requires energy in the form of ATP. Glutamine synthetase simply takes ATP, ammonium and Glu to produce Gln, ADP and Pi . Asparagine synthetase however produces AMP and PPi instead, since the PPi is immediately hydrolyzed by pyrophosphatase the reaction is driven to the products in a similar way as discussed above with argininosuccinate synthetase. Both synthetase reactions are irreversible, hydrolysis of Gln and Asn to Glu and Asp is catalyzed by separate enzymes, glutaminase and asparaginase, respectively.

22.3.3. Gly, Thr and Ser The metabolism of Gly, Thr and Ser are connected, as seen in figure 22.7. From the medical point of view, the following issues are of particular importance: • The reactions from Thr to Gly or succinyl-CoA are irreversible, making Thr an essential amino acid. • Deficiency of glycine cleavage enzyme leads to non-ketotic hyperglycinemia, a rare (1:250 000) autosomal recessive disorder of amino acid metabolism. It results in severe, often fatal, neuronal deficiencies. • Excessive conversion of glycine to oxalate can lead to kidney stones, since Ca-oxalate has a low solubility in water. In such cases restriction of Gly in the diet is the best treatment. • Trimethylamine causes the smell of “ripe” fish. It’s N-oxide is used as osmolyte by fishes, after their death bacterial degradation leads back to the amine. • The pathways involve two carriers of C1-bodies, SAM and folate. These will be discussed in the next subsections.

418

22.3.3

Gly, Thr and Ser

Figure 22.7.: Threonine can be converted to succinyl-CoA, pyruvate or glycine, these reactions are irreversible making Thr an essential amino acid. Gly can be converted to oxalate, trimethylamine, Ser, or cleaved to CO2 and water. Several of these reactions require C1-carriers, either SAM (see section on Met) or THF. COO

-

COO

-

COO H3N

NADH 2 H+

Oxalate

NAD+ H2O

kidney stones with Ca2+

-

Tetrahydrofolate (Vit. M)

FADH2 NH4+

Glyoxylate

Ser hydroxymethyl transferase Methylene THF + H2O

B6 FAD H2O

2 SAH

CH

Peroxisomes

CHO

CO2

-

C OH H2

COO

Bacteria

+

COO

2 SAM

+

HN (CH3)3

H3N

Trimethylammonium-ion

+

-

B6

CH2

CO2 + NH4+

glycine cleavage enzyme

Glycine

-

Methylene-THF

THF

recessive deficiency (1:250,000) non-ketotic hyperglycinaemia fatal or severe mental deficiency

OH

Acetyl-CoA

H2O N(CH3)3

CoA-SH

Trimethylamine

COO

[O]

H3N

+

CH3 O

N CH3

COO

CH

-

C O

C O

CH3

CH3

α-amino-β-ketobutyrate

NADH

CH3

-

Pyruvate

Trimethylamine-N-oxide NAD+

COO H3N

+

-

CH HC OH CH3

Threonine

NH4+ B6 threonine dehydratase

COO

-

O C CH2 CH3 α-ketobutyrate

CoA-SH NAD+

CO2 NADH

B1 α-ketobutyrate dehydrogenase

O C S CoA CH2 CH3 Propionyl-CoA

Succinyl-CoA

419

22.3.3

Biochemistry and Genetics

Figure 22.8.: Folate (Vit. M) is required for the synthesis of nucleic acids. Several important drugs interfere with folate metabolism. N

H2N

H

N

HN

N

O

CH2

O

N H

Pteridine derivative

para-Amino-benzoic acid

H2N

O COOH C N CH H CH2

S NH2 O

Sulfanilamide (Antibiotic)

CH2

Folic acid, Viatamin M

COOH Glutamic acid

H H2N

N 1

2

HN

3 4

NADPH+H+

H

N 8

NADP+

H2N

7 6

5

N

O

10

Folate

HN

Dihydrofolate reductase

CH2

H

N

H

HN

7,8-dihydrofolate

H N

N

H

NADPH+H+

H CH2

O

N R H

H2N

N

N

NADP+

N

H2N

N O

R

H

H H

HN

Dihydrofolate reductase

N

CH2

H

HN

5,6,7,8-tetrahydrofolate

R

H H

HN

N O

CH2

N H R HC

Gly metabolism

NH N5-formimino-THF (from His-catabolism) Formimino-THF cyclodeaminase

NH3 H H2N

N

H

N

H H

NADPH + H+ NADP+

H

HN

N O

+

N

O

R

N

H

H NADH + H+

H

NAD+

N

CH2

C H2

N

(Formaldehyde, dUMP -> dTMP)

H H2N

N

N

H

H

HN

N O

ATP

H2N

CH2

N H R HC

N10-formyl-THF isomerase

N

N

H

HC

(formic acid, purine synthesis, formyl-Met-tRNA synthesis)

420

OMe

HN R O

N5-formyl-THF

Trimethoprim (DHF reductase inhibitor, bacteriostatic)

H CH2

O N10-formyl-THF

OMe

NH2

N O

N N

H

HN

R

(Methanol, Homocysteine -> Methionine)

H2 N

H H

CH2

CH3 HN

OMe

N5-formyl-THF cycloligase

Methenyl-THF cyclohydrolase

H

N5-methyl-tetrahydrofolate

ADP + Pi H2O

H

N O

R

N

H

HN

N5,N10-methylene-THF

N5,N10-methenyl-THF

N

H2N

H

HN

CH2

C H

N

H2N

N

H2N N

NH2

N

H

N

CH2 N H3C

Methotrexate (Dihydrofolate reductase inhibitor cancer, arthritis)

O COOH C N CH H CH2 CH2 COOH

Gly, Thr and Ser

22.3.4

Folate as C1-carrier Humans can not synthesize folate, it is an essential nutrient (vitamin M). Dihydrofolate reductase converts dietary folate into its physiologically active form, 5,6,7,8-tetrahydrofolate. THF is used as one carrier of C1-bodies in metabolism. Such C1-bodies originate from amino acid metabolism and are required for the synthesis of nucleotides and, in prokaryotes, formyl-Met. You will recall that in prokaryotes protein starts with an N-terminal formyl-Met, rather than Met as in eukaryotes. The conversion between the various oxidation states of the C1 (methyl-, methylene-, methenyl, formyl- and formimido-) looks daunting, but all you really have to remember is that these reactions are reversible, so that any form produced can be converted into any form required. Because nucleotide synthesis and hence cell division can not occur without THF, several pharmaceuticals have been developed that interfere with folate metabolism and can be used against cancer and/or bacterial infections: Sulphonamides were the first successful broad spectrum antibiotics. The antibiotic effect of Prontosil rubrum® (Sulfamidochrysoidin) was discovered by G.J.P. Domagk in a screen for substances active against wound infections, which had killed many of his contemporaries in the trenches of WWI. The first patient he treated with it was his own daughter, suffering from a gangrenous sports injury. Her successful treatment from a disease which otherwise would have cost her at least the arm, if not her life, started an entire new era of medicine. For his discovery Domagk was awarded the Nobel price for Physiology and Medicine in 1939. He was forbidden from accepting the price by A. Hitler and even imprisoned. Only in 1947 was he able to accept the price (the price money however was returned to the Nobel foundation, since it was not claimed within one year). It was later discovered that Prontosil is a pro-drug, which is converted to sulfanilamide in our body. Since humans can not make folate, sulfanilamide has no effect on human folate metabolism. In bacteria however it stops the synthesis of folate because of its similarity to p-aminobenzoic acid, one of the precursors of folate (see fig. 22.8 for the structures). Chemically modified sulphonamides are also used to inhibit carboanhydrase as diuretics and to lower the pressure inside the eyes. Trimethoprim inhibits dihydrofolate reductase, the enzyme which converts folate into THF. The drug is often given in combination with sulphonamides to prevent drug resistance. Methotrexate (MTX) is another inhibitor of dihydrofolate reductase. It is used to treat malignancies and (in much lower doses) autoimmune-diseases.

421

22.3.5

Biochemistry and Genetics

22.3.4. The sulphur-containing amino acids: Met and Cys Met is required not only for protein synthesis, but also to make S-adenosyl methionine (SAM), the second important carrier of C1-bodies in metabolism (see fig. 22.9). Removal of the methyl-group produces S-adenosyl homocysteine. Removal of the nucleoside from SAH leads to homocysteine, from which methionine can be regenerated using a methylgroup transferred from methyl-THF. If not enough folate is available in the body, homocysteine accumulates leading to vascular complications. Excess folate can mask the pernicious anemia that results from vitamin B12 deficiency, but not its neurological consequences. Since in the absence of anemia hypovitaminosis B12 is much harder to diagnose most developed countries have imposed upper limits on the folate content of vitamin supplements. If more homocysteine is produced than needs to be converted back to Met, it can be condensed with Ser to form cystathionine. This reaction is catalyzed by cystathionine synthase, which is defect in 1:250 000 live births. This autosomal recessive defect leads to homocysteinuria, a disease that leads to mental retardation, dislocation of the eye lenses, bone elongation and osteoporosis and thrombosis. In many cases the enzyme has a lowered affinity for vitamin B6 , the disease can then be treated by very high doses of this vitamin. Cystathionine is broken down to Cys and α-ketobutyrate, the latter being oxidatively decarboxylated to propionyl-CoA. Since Cys can be produced from Met via cystathionine it is not an essential amino acid. Excess Cys is broken down to pyruvate, Cys (and Met) are therefore glucogenic.

22.3.5. Branched-chain amino acids: Val, Ile, Leu All three branched chain amino acids are initially catabolized in a similar way. They are transaminated in muscle by branched chain amino acid transaminase, the resulting αketoacids are transported into liver mitochondria, where they are oxidatively decarboxylated by branched chain α-ketoacid dehydrogenase in a reaction similar to the one you studied for pyruvate dehydrogenase. A defect in branched chain α-ketoacid dehydrogenase leads to maple syrup urine disease, so called because the urine has the characteristic smell of maple syrup, caused by sotolone, a break down product of Leu which by itself is quite harmless. Note that consumption of curry or fenugreek can also lead to sotolone in urine, please avoid prescribing costly laboratory investigations in such cases. Maple syrup urine disease has autosomal recessive inheritance and a prevalence of 1:200 000. Prognosis is very poor, the disease leads to severe mental deficiency, optic atrophy, ataxia, ADHS, axial hypotonia, exertional fatigue, metabolic acidosis, hypoglycemia, elevated liver

422

22.3.5

Branched-chain amino acids: Val, Ile, Leu

Figure 22.9.: A derivative of Met, S-adenosyl methionine (SAM), is the second carrier of C1-bodies in metabolism. NH2

CH3 S

PPi + Pi

ATP

CH2

S

CH2 HC H3N COO

N

N

CH3

H2 C

+

N

N

O

CH2

+

CH2

-

HC H3N

Methionine

COO

+

-

Folate deficiency increases [homocysteine] -> vascular disease

THF

OH

OH

S-adenosyl methionine (SAM)

B12 Methyl-THF

[-CH3]

SH Ado

CH2

S Ado

H2O

CH2

CH2 HC H3N COO

CH2

+

Homocysteineuria 1:200 000 recessive, homocysteine accumulation results in mental retardation, lens dislocation, bone elongation, osteoporosis and thrombosis

Homocysteine

Serine

+

HC H3N

-

COO

-

S-adenosyl homocysteine (SAH)

B6 Cystathione synthase Treatment: Met restriction, mega-doses of B6

H2O

NAD+ CoA-SH

S +

HC H3N COO

CH3

CH2

H2C -

CH2

+

HC H3N COO

-

B1

NADH + H+ CH3 CO2 CH2 O C S CoA

CH2

H2O

O C +NH 4

B6

Propionyl-CoA

COO

-

α-ketobutyrate

Cystathionine

γ-Cystathioninase

COO H3N

+

CH

-

COO

[O2] +

H3N

CH

NH4

+

2-

SO3

COO

-

O C

CH2

CH2

SH

SO2

Cysteine

-

-

Cysteinesulfinate

CH3 Pyruvate

423

22.3.5

Biochemistry and Genetics

Figure 22.10.: Metabolism of branched-chain amino acids. -

COO H 3N

+

B6

CH HC CH3

CO2 NADH

CoA-SH NAD+

-

COO

Glu

αKG

B1

O C

O C S CoA

HC CH3

muscle

CH2

CH2

HC CH3

liver mitochondria

CH2

Valine

Isobutyryl-CoA α-Ketoisovalerate

FAD FADH2

-

COO

NADH + H+

HC CH3 HC

CoA-SH

NAD+

H2O

H2O

O

NAD+ CoA-SH

C CH3

hydration

Oxidation of OH

Methylmalonate semialdehyde

O C S CoA CH2

thioester hydrolysis

Methylacrylyl-CoA B1

NADH CO2

NAD+ CoA-SH

NADH CO2

O

B1

C S CoA

Met

-

COO CO

CH2

CH2

CH3

CH3

Thr

Propionyl-CoA

Remember: this pathway blocked in methylmalonic aciduria

NADH + H+ NAD+

O C S CoA CH2

H2O

-

COO H 3N

+

CH2 -

HC CH3 CH2 CH3

CH3

Tiglyl-CoA

Branched chain a-ketoacid dehydrogenase defect in maple syrup urine disease Prevalence 1:200,000, autosomal recessive Severe mental deficiency, optic atrophy, ataxia, AHDS, axial hypotonia, exertional fatigue, metabolic acidosis, hypoglycemia, elevated liver enzymes elevated branched chain aa in serum, abdominal pain, death in early childhood

H 3N

CH CH2 HC CH3

CH3 Leucine

424

+

2 H ADP

H 2O ATP

-

COO

Succinyl-CoA

C CH3 CH

Isoleucine

+

COO

O C S CoA

CH

O C S CoA

Pi

CO2

CH

CH C CH3 CH3 methylcrotonyl-CoA

O C S CoA

Biotin

C CH3 CH2

-

COO

Methylglutaryl-CoA

H 2O HMG-CoA

Phe and Tyr

22.3.6

enzymes and branched chain amino acid concentrations in serum and abdominal pain. Death occurs usually in early childhood. Val and Ile are finally broken down to propionyl-CoA, Leu to HMG-CoA. Note that the conversion of propionyl-CoA to succinyl-CoA is defect in methylmalonic aciduria. These patients therefore have not only problems with fatty acid metabolism, but also with metabolism of Val, Ile, Met and Thr.

22.3.6. Phe and Tyr The metabolism of Phe and Tyr contains a number of important inherited diseases, which are frequent topics in board exams.

Phenylketonuria and albinism Phe is converted to Tyr by phenylalanine hydroxylase, a mixed oxygenase. One of the atoms of an oxygen molecule is inserted into the para-position of Phe, the other oxidizes tetrahydrobiopterine (BioH4 ) to dihydrobiopterine (BioH2 ). The latter needs to be converted back to BioH4 by dihydrobiopterine reductase. An inherited recessive defect in either phenylalanine hydroxylase (1:200 000 in most populations, but up to 1:7000 in people of European descent) or dihydrobiopterine reductase (much rarer) results in PKU, a serious medical condition that leads to seizures, spasticity and irreversible brain damage. Treatment is by Phe restriction until after adolescence, when the brain is fully formed and becomes less sensitive to the breakdown products of excess Phe. Cave: Aspartam contains Phe and Tyr becomes essential. To avoid brain damage the law in most developed countries requires mandatory testing for PKU in newborns (for newborn screening, see section 28.1.3 on page 513). Defects in dihydrobiopterine reductase also prevent the production of l -DOPA and 5-OHTrp. This affects the serotonine and catecholamine metabolism (see chapter ?? on page ?? for a discussion of hormone synthesis). In occulocutaneous albinism the synthesis of melanin (dark pigment of skin, hair and iris) and pheomelanine (pigment of red hair) is defect. This results in white hair and skin and in a red iris (because the retina shines through the unpigmented iris). Affected persons do not have the protection from UV-radiation which the presence of melanin provides. As a result they suffer from sunburns easily and may develop skin cancer. The only known treatment is to totally avoid exposure to the sun.

425

22.3.6

Biochemistry and Genetics

Figure 22.11.: Metabolism of Phe and Tyr. NAD+

NADH + H+

Rare form of PKU: Inability to synthesise L-dopa and 5-OH tryptophane. These intermediates must be supplied in the diet.

Dihydrobiopterine reductase

H N

N

H2N HN

O

N

HN H H C C CH3

N H

Note: same enzyme used in Tyr- and Trp-hydroxylase: and catecholamine H Serotonine N biosynthesis also affected

HN

H H C C CH3

N

OH OH

O

Tetrahydrobiopterine (BioH4)

OH OH

Phenylalanine hydroxylase

+

O2

+

(PKU): autosomal COO Phenylketoneuria recessive, 1:6 000 (UK) to 1 : 200 000

COO

CH (Japan). Mental retardation, seizures,

+

H3N

spaticity. Mandatory diaper test in most

CH2 developed countries. Treatment by Phe

restriction until after adolescence. Cave: Aspartame! Tyr becomes essential.

Hawkinsinuria: very rare. Acidemia and excretion of hawkinsin. Tyr-restricted diet during first year of live. Autosomal dominant. Alkaptoneuria: excretion of homogentisate in urine (-> black). Ochronosis (pigment accumulation in tissue), arthritis.

COO

COO

CH2

H+

O OOC

HO

COO O H

O

OOC H

Furmarylacetoacetate

Tyrosine aminotransferase

-

CH2

OH pyruvate oxidase

2

-

H2O

O C

O2

OH

Tyrosinaemia type I: cabbage-like smell from FAA, liver + kidney failure, liver cancer. 1 : 100 000, but 1 : 2 000 in some areas of Quebec H+ HO

CH2

Maleoylacetoacetate isomerase

OH

Tyrosinaemia type II: < 1 : 250 000, affects brain, eyes and skin.

COO

Homogentisate

-

+

p-hydroxyphenyl

Homogentisate

H

CH2

C

O 1,2-dioxigenase

Maleoylacetoacetate

426

CO2

Pheomelanine

CH

Glu

Nitisinone, NTBC

CH2

O2

-

H

-

Dopa

-

αKG

Tyrosinaenmia type III: very rare, mild mental retardation, ataxia

-

Dopamin

Occulocutaneous albinism

-

H3N

Melanin

Dihydrobiopterine (BioH2)

Fumarylacetoacetate hydrolase

Hydroxyphenyl pyruvate

COO

+

CH CH COO Fumarate

COO

-

-

-

CH2 CO CH3 Acetoacetate

Trp and Lys

22.3.7

Tyrosinemia and Alkaptonuria The breakdown of Tyr is associated with several autosomal recessive deficiencies, the most serious of those is tyrosinemia type I. The disease results from a deficiency of fumarylacetoacetate hydrolase which results in a cabbage-like body smell from FAA, liver + kidney failure, liver cancer. The frequency is about 1:100 000 in most populations, but because of a founder effect 1:2000 in some areas of Quebec (Canadian students beware!). Tyrosinemia II and -III are much less serious than type I. Type II results from a deficiency in tyrosine aminotransferase. The disease has a frequency of 1:200 000 and results in damage to brain, eyes and skin. Type III results from a defect in p-hydroxyphenyl pyruvate oxidase and is very rare. It causes mild mental deficiencies and ataxia. A very rare partial defect in PHPO leads to hawkinsinuria: The enzyme produces a reactive intermediate, 1,2-epoxyphenyl acetic acid instead of homogentisate. The intermediate then reacts spontaneously (epoxy-group!) with glutathione to produce hawkinsine. Because the production of intermediate is a gain-offunction mutation, hawkinsinuria has dominant inheritance, which is rare for metabolic diseases. Fortunately, the disease is relatively benign, leading to acidemia only in the first year of life (treat with Phe + Tyr-restricted diet). Afterwards, patients become quite normal except for the excretion of hawkinsin, which gives the urine a chlorine-like smell. Alkaptonuria is almost benign, except for an increased risk for arthritis later in life. However, the deficiency of homogentisate oxidase leads to the excretion of homogentisate in the urine, which forms a dark pigment when exposed to air. The pitch-black diapers tent to alarm the parents, who will come storming into your surgery. Simply tell them that you know what it is and that it is not dangerous. A different pigment will over the years accumulate in the tissue of the patient, this is called ochronosis. There is no treatment for this “disease”.

22.3.7. Trp and Lys Catabolism of Trp starts by opening the 5-membered ring with a dioxygenase, via a couple of steps this part of the molecule is converted to Ala. Then the 6-membered ring is opened and converted via α-ketoadipate to acetoacetate. There are a couple of things to remember about the catabolism of Trp: • Xantenurate and kynurenate originate from this pathway, they cause the yellow color of urine. • Trp can be converted via quinolinate into nicotinamide (vitamin B3 ). The process is not very efficient, about 60 mg of Trp need to be catabolized to produce 1 mg of

427

22.3.7

Biochemistry and Genetics

Figure 22.12.: Metabolism of Trp and Lys. +

H2 N H3 C C COO H

+

H2 N H3 C C COO H H C N O H2O H

O2

O

Tryptophan dioxygenase

N H

N-formylkynurenine

Tryptophan

Formamidase

Xantenurate (yellow colour of urine)

H2O

+

B1

Kynureninase

COO

Kynurenine-3monoxygenase

NH2 -

OH

O2

O C H

NH2 3-HA-3,4-dioxygenase OH

COO -

OOC

+

O2 NADPH

NADP+ H2 N H3 C C COO H

O

H2O Alanine

HCOO-

OH

NH2

N

Kynurenine

COO spontaneous

NH2

N

COO

-

-

Quinolinate (precursor of NAD) CO2

NAD+ NADH

H2O

NAD(P)H NAD(P)+ NH3

COO O

S CoA

C

CH2

Acetoacetate

NADH CO2

NAD+ CoA-SH

CH2

B1

CH2 CH2

COO

-

α-Ketoadipate

COO

Glutaryl-CoA

COO H3N

CH CH2

NADPH

COO NADP+

CH2 CH2 CH2 +

N H3 Lysine

428

aminoadipate aminotransferase

-

α-KG

Saccharopine dehydrogenase (lysine forming)

H3N

+

B6 α-KG

-

CH CH2

COO

CH2

CH2

CH2

CH2

-

N H

COO

Saccharopine

NADH

COO +

Glutamate H3N

CH

-

COO NAD(P)+

NAD(P)H

Saccharopine dehydrogenase (glutamate forming) -

CH2 CH2 HC

O

α-Aminoadipate-6semialdehyde

+

H3N

-

CH CH2

CH2

CH

H2C

H2O NAD+

-

Glu

Hyperlysineaemia/-uria: mental and physical retardation +

-

O C

CH2 CH2

COO

Kynurenate (yellow colour of urine)

H2O

-

2-Amino-3-carboxy muconate6-semialdehyde

3-Hydroxyanthanilate

H2 N H3 C C COO H

O

aminoadipatesemialdehyde dehydrogenase

CH2 CH2 COO

-

α-Aminoadipate

-

Pro, Arg, His and ornithine

22.3.8

nicotinamide. Since proteins on average contain about 1 % Trp about 6 g of protein are required per mg of nicotinamide. Lys is converted in liver mitochondria first to α-ketoadipate, then the pathway to acetoacetate is the same as for Trp. Since all reactions from Lys to α-ketoadipate are reversible, it is possible to make small amounts of Lys from Trp in our body. However, this is insufficient to meet our Lys-needs, making Lys an essential amino acid. There is a rare deficiency associated with the first two steps of Lys-degradation, hyperlysinemia/uria, which results in impaired sexual development, muscular and ligamentous asthenia (weakness), normocytic, normochromic anemia, convulsions, episodic vomiting, rigidity and in extreme cases coma. The disease locus is 7q31.3, in humans both saccharopine dehydrogenase activities are located in the same, bifunctional protein [Sacksteder et al., 2000]. Apparently, excess Lys is a competitive inhibitor of arginase, resulting in hyperammonemia, which is toxic. Treatment is by low protein diet if the disease is severe enough to warrant that. In brain peroxisomes a second pathway for Lys degradation exists, however, no medical conditions have been linked to it and we will not discuss it any further.

22.3.8. Pro, Arg, His and ornithine Pro, Arg and His are all catabolized to Glu, which can then enter the Krebs-cycle as discussed above. The pathway from Pro is reversible, thus we can produce Pro from Glu and Pro is not essential. Note however that the conversion between ∆5 -dehydroproline and Pro is catalyzed by different enzymes, the catabolic enzyme is a flavoprotein, the anabolic uses NADPH + H+ . Excess Arg is converted via the urea cycle to ornithine, which is converted to Glu. Since the reactions involved are reversible ornithine can be made from Glu should the flow through the urea cycle need to be increased. The breakdown of His to Glu is not reversible, making His an essential amino acid. However, there is so much carnosine (βAla-His) in our muscles that His-deficiency in the diet can be masked for a long time by carnosine breakdown. Therefore, no dietary requirement for His could be established. There are two inherited deficiencies associated with His-catabolism, both are completely benign. However, they may interfere with laboratory procedures, so you have to know about them: Histidinemia is caused by a deficiency in histidinase, the first enzyme in the pathway. The excess His in the blood and urine does not cause health problems, but does react with FeCl3 in the same way as Phe, giving a false positive diaper test in the screen for

429

22.3.8

Biochemistry and Genetics

Figure 22.13.: Pathway for the degradation of Pro, His, Arg and ornithine. COO H3N

-

+ H2N

COO H

+

+

H3N

CH (CH2)3 NH

H2N

Proline

COO

+

C

β-Ala-His (Carnosine)

urea

COO +

H3N

-

COO H

N

-

defect 1:10,000 -> histidinaemia benign, but false positive phenylketoneuria test Diagnostic: Lack of Urocanate in sweat.

CH

-

CH

N

ornithine

Urocanate

H2O

H2O

spontaneous

αKG

COO H3N

COO

N

NH2

+

+

N H4

CH

(CH2)3

N Δ5-dehydroproline

N

histidinase urea cycle

NADPH + 2 H+

FADH2 H+

CH2

histidine

H2O

NADP+

CH

NH2

arginine FAD

-

-

Glu

CH

H2O

(CH2)2

H+

CH O

COO

glutamate-γ-semialdehyde NAD+

CH2 CH2

+ ADP + Pi

N NADH + H+ + ATP

COO H3N

+

-

glutamate

430

THF

-

Formimino-THF Test for folate deficiency: FIGLU in urine after oral His load

-

N-Formiminoglutamate (FIGLU)

Enzyme Deficiency: habitual excretion of FIGLU in urine: benign

(CH2)2

COO NH

glutamate formimino transferase

CH COO

-

22.4.2

Carnitine

Figure 22.14.: Synthesis of carnitine COO H3N

+

-

CH CH2

COO [O]

H3N

+

CH

H O C

Glycine

HO CH

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

N

+

H3C

-

N

CH CH3 3

H3C

N

CH2

H3C

+

CH CH3 3

+

CH CH3 3 NAD+

ε-N-Trimethyllysine

NADH

COO

-

CH2

COO [O]

CH2 CH2

HO CH

H3C

CH2

CH2

N

N

+

CH CH3 3

-

H3C

+

CH CH3 3

Carnitine

PKU. Patients showing a positive screening result therefore need to be investigated by more selective, but also much more complicated and expensive, methods. Habitual FIGLU excretion occurs when the enzyme catalyzing the last step of the pathway is defect. Usually this totally benign condition is not even noticed. However, the appearance of FIGLU in the urine after a challenge with a large dose of His is used to diagnose folate deficiency. Again, the enzyme deficiency would cause a false positive result if the absence of FIGLU in urine were not confirmed before His is given.

22.4. Compounds derived from amino acids 22.4.1. Carnitine Carnitine is required for fatty acid metabolism. It can be synthesized from Lys, and is therefore not an essential ingredient of food. Supplementation with carnitine is not required even for physically active persons.

431

22.4.3

Biochemistry and Genetics

Figure 22.15.: Creatine as energy storage molecule in muscle. from Arg

H2N

+

from Gly from SAM

2-

NH2

ATP

C N C COO H CH3 2

ADP

-

creatine kinase

+

H2N

HN PO3

N C COO H CH3 2 Creatine phosphate

Creatine

this enzyme is found in serum after damage to muscle or heart

Pi

C

HN C -

spontaneous

H N C O N

CH2

CH3 Creatinine this substance is used to measure renal (glomerular) activity

22.4.2. Creatine and creatinine Sudden muscular activity in flight-or-fight response requires energy in form of ATP. However, only small amounts of ATP can be stored inside a cell. Muscle cells therefore store energy in the form of creatine phosphate, a super-energy rich compound (∆G00 = 43 kJ/mol) which is readily converted back to ATP and creatine. Creatine itself is synthesized in our body from Arg, Gly and SAM. A small percentage of the creatine phosphate stored constantly breaks down into creatinine, this is given into the blood stream and excreted by the kidney. There is no reabsorbtion of creatinine in the kidney, all creatinine filtered by the glomerulus into the primary urine therefore ends in the urine. The ratio of creatinine concentrations in urine and plasma multiplied by the urine production rate therefore gives the volume of blood filtered by the kidney per unit time (glomerular filtration rate (GFR)), which is used clinically to assess kidney function. The enzyme responsible which catalyzes the conversion of creatine to creatine phosphate is creatine kinase, a cytosolic enzyme which is released into the blood stream when the cell is damaged. It is easily measured by the coupled spectrophotometric test of Warburg (see section 7.9 on page 169) and can be used to help in the diagnosis of myocardial infarct and myopathy (see section 10.4 on page 208).

22.4.3. Polyamines Polyamines are synthesized in our body either by decarboxylation of Lys (cadaverine) or from ornithine (putrescine, spermidine and spermine). The exact function of polyamines has not been established. It is assumed that the regularly spaced positive charges from the amino groups helps packing DNA. There also seems to be a role in the regulation of ion channels, in particular NMDA and AMPA receptors and the inwardly-rectifying potassium channels.

432

22.4.3

Polyamines

Figure 22.16.: Biosynthesis of polyamines NH2

S

N

N

CH3

H2 C

+

N

O

+

H3N

CH2

N

H2C

CH2 CH2

CH2 + H3N CH COO

+

HC H3N COO

OH

-

OH

Ornitine S-adenosyl methionine (SAM)

Ornitine decarboxylase

SAM decarboxylase

CO2

CO2

NH2 CH3 S

+

O

N

CH2

H2N

C O

H2C

N

CH2 H2C

CH2 CH2

H3C

N

N H2 C

+

H3N

H2O2

Putrescine

+

CH2 H2C CH

+

H3N

O

+

C H3N OH H2

O2

OH polyamine oxidase

H2O

+

H3N

H3C S Ado

CH2 +

H2C H2N

H2C

CH2

CH2

H2C

AcetylCoA

C O

CH2

+

CH2 H2C + H2N H2C CH2 H2C + H3N

H3C

H3N

CH2

H2N

Spermidine diamine-N-acetyl transferase

H2C Spermine

+

AcetylCoA

diamine-N-acetyl transferase

CoA-SH

CH2

+

H2N

H2C

H3N

CoA-SH

CH2 H2C + H3N

polyamine oxidase

O H3C

C

+

N H2

H2 C

C H2

H2 C

+

N H2

N1-acetyl

H2 C

C H2

H2 C

spermine

+

N C H2 C H2 H2

N1-acetyl spermidine

H2C

CH2

CH2 H2C

+

CH2

+

CH2

H3C S Ado

H2N

H2 C

+

N C H3 H2

H3N H2O2

+

CH2 H2C CH

O2

O

H2O

433

22.4.3

Biochemistry and Genetics

Figure 22.17.: Mechanism of the drug Eflornithine. Difluoromethylornitine

O Pi

+

H2 O C

OH N

+

+

N H3

CH3

CHF2

C C C C COO H2 H2 H2 + H3N

N H3

CH H C C C C COO H2 H2 H2 + H3N

H2O

PLP

CHF2

H2O

+

N H3

N

-

C C C C COO H2 H2 H2 + H N CH H2 OH P O C

N H3

H C C C C COO H2 H2 H2 + H N CH H2 OH P O C

+

-

N

CH3

CH3 CO2 F CHF +

CO2

+

H C C C C H2 H2 H2 + H N CH2 H2 P O C

N H3

N H3

N

C C C C H2 H2 H2 H N CH2 H2 P O C

Enzyme-Cys-SH

OH

N

OH CH3

CH3 HF Enzyme-S

H2O

CH +

N H3

O H2 P O C

CH OH N

434

CH3

+

+

N H3

+

C C C C H3N H2 H2 H2 H2

C C C C H2 H2 H2 + H N CH2 H2 P O C

N

OH CH3

Inherited amino acid transporter deficiencies

22.5.2

Inhibition of polyamine synthesis by inactivation of ornithine decarboxylase with difluoromethylornithine (Eflornithine® ) is used to treat cancer, seizures and sleeping sickness, an infection caused by Trypanosoma brucei. Especially in the latter role Eflornithine® has become known as the “resurrection drug”, since it can be used to treat even the second, meningoencephalitic, phase of the disease. Because Eflornithine® blocks only the synthesis, but not the uptake, of polyamines, its effectiveness in treating cancer was found to be limited. Compounds that stimulate polyamine oxidase, and hence the destruction of polyamines (synthesized by the cell or taken up), are currently in clinical trials.

22.5. Physiology of amino acids 22.5.1. Metabolism of amino acids Most amino acid catabolism occurs in the liver. However, branched chain amino acids are transaminated in muscle, the corresponding α-ketoacids are then transported to the liver for further metabolism. Ala is produced in the anaerobic muscle and transported to the liver via the blood stream. Thus rather than transporting two potentially dangerous metabolites – lactate and ammonia – only the relatively harmless Ala needs to be transported. In the liver the nitrogen is disposed of by the urea cycle, while the pyruvate is used for gluconeogenesis. This is called the Ala-cycle.

Glu + Gln form the major energy source for the intestinal mucosa. Gln is required for the metabolic compensation of acidosis by the kidney: Glutaminase and glutamate dehydrogenase produce ammonia, which binds a proton to form ammonium that ends up in urine. In addition, gluconeogenesis from α-ketoglutarate binds another 4 protons: 2 αKG + 4 H2 O + 4 H+ → Glc + 4 CO2 + 8 [H]. This reaction however occurs also in starvation and exertion, as the kidney is as important for gluconeogenesis as the liver. The Gln-Glc cycle between muscle and kidney corresponds to the Ala-cycle between muscle and liver.

22.5.2. Inherited amino acid transporter deficiencies Cystinuria is caused by a defect of the transporter for the dibasic amino acids Lys, Arg, cystine (Cys-S-S-Cys) and ornithine in the intestine and in the kidney. Intestinal uptake of these amino acids in the form of di- and tripeptides is usually sufficient to meet the dietary needs of the patient (especially in rich countries with high dietary protein content) but failure of cystine-reuptake in the kidney leads to cystine-concentrations in the urine which exceed its solubility. This results in the formation of kidney and bladder stones. Patients

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Biochemistry and Genetics

should maintain a large urine volume and a slightly alkaline urine pH (≥ 8.5) to prevent the crystallization of cystine. The transporter has two subunits, a large (SLC3A1 on 2p16.3, type A cystinuria) and a small (SLC7A9 or rBAT on 19p13.1, type B cystinuria). Mutations in both subunits are called type AB. Depending on the mutation inheritance can be recessive (type I) or incomplete recessive (type II and III, heterozygotes show moderate or low increases of dibasic amino acids in urine, respectively). It is estimated that about 10 % of stone formers are heterozygous. The overall frequency of cystinuria in the population is 1:7000, making it one of the most prevalent genetic disorders. Hartnup disease is caused by a defect in the transporter for large neutral amino acids (SLC6A19 on 5p15). This results in a relative Trp-deficiency, making the patient more susceptible to pellagra. There may also be a (usually mild) encephalopathy due to insufficient myelin-formation. In industrialized nations the signs of the disease are rarely seen because of the super-adequate diet. The frequency is reported to be between 1:14 000 and 1:24 000. Oasthouse disease is caused by a defect in Met-uptake in the intestine. As a consequence, Met is metabolized by intestinal bacteria to α-hydroxybutyrate (do not mix up with the ketone body β-hydroxybutyrate!). This compound is absorbed in the intestine, but not metabolized in the body. Rather, it is excreted by the kidneys, giving the urine a characteristic smell of drying hops (hence the name of the disease). Patients have striking white hair, suffer from mental retardation, convulsions, tachypnea and diarrhea. The disease is inherited in an autosomal recessive pattern, and is exceedingly rare.

22.6. Exercises 1. Protein consumption The RDA for protein intake is 56 g/d, average uptake in US 34 g/d plant and 75 g/d animal protein. Metabolism of the excess amino acids places additional strain on liver and kidney, worsens metabolic syndrome and may cause gout. A diet high in (animal) protein results in an overdose of saturated fat but lacks fibre. Feeding livestock requires 40 % of the worlds grain and 95 % of its soy production, 41 × 106 t of plant protein are required to produce the 7 × 106 t/a of animal protein consumed in the US. 2/3 of worlds arable land is used to produce feed stuff and 87 % of its water (100 000 l per kg of beef). Livestock in US produces 1.4 × 106 t/a of manure, 130 times the human production. Production of methane, ammonia and nitrous oxide by cattle is a significant contributor to acid rain and global warming. Fuel consumption per kg protein is 10 times higher for beef than plants. Thus the consumption of excess animal proteins in rich countries contributes

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Objectives in Summary: Amino acid metabolism

22.7

to health and environmental problems and to the hunger in developing countries. With consumption of animal proteins increasing in India and China (which between them have about 1/3 of the worlds population) these problems are likely to increase in the future. Question: If all animal protein consumed by the 300 × 106 citizens of the US were replaced by plant protein, and protein uptake restricted to recommended doses, approximately how many additional people could be fed? A 100 million B 200 million C 500 million D 1 billion E 2 billion 2. Biological value of proteins Calculate the protein score for a mixture of 2 parts rice and 1 part soy beans, using the composition data provided above. Which is the limiting amino acid? A Ile B Lys C Met D Phe E Val

22.7. Objectives in Summary: Amino acid metabolism At the end of this lecture students should be able to • distinguish between essential and non-essential amino acids • describe how the amino acid pool of our body is filled and used and define the terms “nitrogen balance” and “biological value”. • advice patients on nutrition with respect to proteins. • describe how the amino acid nitrogen is used and eliminated in metabolism. • describe how the carbon chains of amino acids enter major metabolic pathways. • define the role of alcohol in this context.

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Biochemistry and Genetics

• describe the role of enzymes of amino acid metabolism in laboratory medicine. • describe the role of folate in metabolism and give example for pharmaceuticals that interfere with it’s function. • describe the consequences of hypovitaminosis M and B12 and the interaction between these nutrients. • describe non-nutritional functions of amino acids. • describe the role of polyamines in health and disease and the pharmaceutical use of difluoromethylornithine and polyamine oxidase activators. • describe the patho-mechanism, signs and symptoms, treatment options and probable outcome of – urea cycle failures – non-ketotic hyperglycemia – histidinase deficiency – homocysteinuria – maple-syrup urine disease – methylmalonic aciduria – phenylketonuria – tyrosinemia type I, II and III – hawkinsinuria – alcaptonuria – hyperlysinemia – cystinuria – cystinosis – oasthouse disease – Hartnup’s disease

438

23. Biochemistry of Digestion 23.1. Digestion Most dietary nutrients are macromolecules that have to be hydrolyzed to their building blocks in the digestive tract. The products of digestion are absorbed: Nutrient Enzyme Product Proteins Proteases Amino acids, di- and tripeptides Starch Glycosidases Glucose Disaccharides Glycosidases Monosaccharides Triglycerides Lipases Fatty acids, 2-monoacylglycerol Nucleic acids Nucleases Nucleosides, bases ≈ 30 g of digestive enzymes are secreted/day.

23.1.1. Digestive secretions in man Salivary glands pH of saliva is 6.0–7.0. It contains 3 important enzymes: α-Amylase: Endoglycosidase, cleaves α-1,4 glycosidic bonds in starch and glycogen. Forms maltose, maltotriose, and α-limit dextrins. Quantitatively less important than pancreatic isoenzyme, but keeps teeth clean. Lysozyme: Endoglycosidase, cleaves β-1,4 glycosidic bonds in the bacterial cell wall polysaccharide peptidoglycan. Most Gram-positive bacteria are killed by this enzyme, Gramnegative bacteria are protected by outer membrane. Lysozyme is also present in other body secretions, egg white, lysosomes of phagocytic cells, etc. Wound-licking is an instinctive way to prevent wound infection. lingual lipase digestion of small part of dietary fats These enzymes are inactive at the acidic pH of the stomach.

439

23.1.1

Biochemistry and Genetics

Figure 23.1.: Starch digestion. α-amylase can digest starch to within 6–8 glucose molecules of a branch-point. The resulting limit dextrin is digested by isoamylase. non-reducing end O

non-reducing end O

H

H2C OH H O

HO H H

H H2 C OH H

HO

H

OH O

α(1->4)

H

O HO

H

H2C OH H O HO H H

H

H

H H

OH

HO

H2C OH H O

O

HO H H

OH

Amylase

H H2 C OH H O

HO

O

Amylase

H

O

H

H

HO

H O

H2C OH H O HO H H

OH

H

H H

H H H2 C OH H O

Isoamylase

HO H

H O

H2C

HO H H

O H

α(1->6) O

OH O

H H H2C OH H O

HO H H

H H

OH O

Amylase

HO H H

OH CH2 H O OH

H H H2C OH H O

O

HO H H

440

OH

H O

reducing end

23.1.1

Digestive secretions in man

Figure 23.2.: Structure of murein and its digestion by lysozyme. cleavage site of lysozyme GlcNAc O

NAM

GlcNAc

OH OH β1->4 H2C H2C O O O HO

N H3C

C

O HC

OH CH3

O C l-Ala O

β1->4

NAM

OH O

O H2C HO

N H3C

iso-d-Glu

C

d-Ala

O

OH O

β1->4

O

iso-d-Glu d-Ala Gly5 l-Lys

iso-d-Glu

l-Lys

l-Ala

iso-d-Glu

O C

H2C

O OH H O C CH 3 C l-Ala O

d-Ala Gly5 l-Lys l-Lys

β1->4

d-Ala

l-Ala O C

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23.1.1

Biochemistry and Genetics

Stomach The pH of stomach is 1.5–3.0. Functions of gastric acid: • Antibacterial action • Denaturation of proteins, which facilitates the action of proteolytic enzymes. Pepsin, the major enzyme of the stomach, is an endopeptidase with a pH optimum at 2.0–2.5. Unlike exopeptidases (aminopeptidases and carboxypeptidases) which cleave only terminal peptide bonds, endopeptidases also cleave internal bonds, pepsin after aromatic + large hydrophobic aa. Pepsin produces a mix of oligopeptides (“peptones”). Only small amounts of free amino acids are formed. Total gastrectomy is compatible with life: Mild digestive problems, increased risk of intestinal infections, but vitamin B12 supplements are required. Pancreas The pancreas supplies most of the soluble enzymes in the intestine. These enzymes function at the near-neutral pH of the small intestine. Major secretions: Bicarbonate neutralizes stomach acid, buffers to neutral pH Endopeptidases: The pancreatic endopeptidases are serine proteases (they have a serine in their active site), and they have different cleavage specificities: Trypsin cleaves on the carboxy side of Lys and Arg. Chymotrypsin cleaves on the carboxy side of hydrophobic amino acids. Elastase cleaves on the carboxy side of small amino acids. Carboxypeptidases: The pancreas secretes 2 zinc-containing carboxypeptidases: Carboxypeptidases A cleaves hydrophobic amino acids from the C-terminus. Carboxypeptidase B cleaves basic amino acids (Lys, Arg) from the C-terminus. α-Amylase: Similar to salivary α-amylase. Same cleavage specificity but different molecular structure: The two α-amylases are isoenzymes. The pancreatic enzyme is more important for digestion than the salivary enzyme. Lipase: The pancreatic lipase is the major enzyme of fat digestion. Reaction: Triglyceride → 2-Monoacylglycerol + 2 fatty acids Also required: Co-lipase, a pancreatic protein which anchors the lipase to the surface of fat droplets.

442

Digestive secretions in man

23.1.1

Figure 23.3.: Cholate (PDB-entry 2qo4). All the OH-groups are on one face of the molecule, making it able to interact with water. The other face is hydrophobic (hydrocarbons) and interacts with lipid. Note that hydrogen atoms are not visible in X-ray crystallography.

Bile salts (from liver) Phospholipases and nucleases take care of dietary phospholipids and nucleic acids. Pancreatic failure cause steatorrhea + generalized lipid malabsorption. Risk of deficiencies of fat-soluble vitamins! Liver and bile bladder Bile salts (20–50 g/d) are required for lipid digestion in the small intestine. Like detergents they form mixed micelles which ferry the lipids to the mucosal surface. The break-up of lipid/detergent micelles is aided by peristaltic motion. Absence leads to steatorrhea, e.g. if bile stones block bile duct. The intestinal brush border The lumenal surface of the intestinal mucosal cells (“brush border”) has membrane-bound digestive enzymes: Aminopeptidases complete protein digestion. Considerable amounts of di-and tripeptides are absorbed intact and hydrolyzed by cytoplasmic enzymes in the mucosal cells. Endo- and carboxypeptidases act on oligopeptides Sucrase cleaves sucrose to glucose + fructose. Lactase cleaves lactose to glucose + galactose. Glucoamylase removes glucose from the non-reducing end of starch, starch-derived oligosaccharides and maltose. Isomaltase cleaves α-1,6 bonds in isomaltose and α-limit dextrins.

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Biochemistry and Genetics

Figure 23.4.: Oligosaccharides come in two types: In the trehalose type the C1 of one sugar is linked to the C1 of the other. Those oligosaccharides are non-reducing and show no mutarotation. In oligosaccharides of the maltose type the anomeric the (C1 ) carbon of one sugar is linked to a non-anomeric carbon of the other. This still has a reducing end. Trehalose-type

Maltose-type

H2C HO HO

OH O

O HO

HO HO

OH

H2C

CH2 O

Glc (α1->6) Glc Isomaltose

OH

HO HO OH

HO CH2 O O

OH OH

HO

Glc (α1->α1) Glc Trehalose

hemiacetal (reducing, mutarotation)

O

OH O OH

OH O

HO HO

444

OH

OH

Glc (α1->4) Glc Maltose

HO HO

H2C

OH H2C O

OH

H2C

acetal (non-reducing)

acetal (non-reducing)

OH O

OH

OH

CH OH OH 2

O

HO CH2 Glc (α1->β2) Fru Sucrose

O

Lactose intolerance

23.1.4

Trehalase cleaves trehalose (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside).

23.1.2. Undigestible materials Many plant products, including cellulose, hemicelluloses, lignin etc. are not digested. A small proportion of these (≈5 %) is fermented by colon bacteria forming acids (lactic, acetic, propionic acid) and gas (H2 , CH4 , CO2 ). Carbohydrates which are not digested but rapidly fermented by colon bacteria cause flatulence. Inulin (poly-fructose is the energy store of asteraceae and umbelliferae). Raffinose (gal(α1 → 6)glc(α1 → 2)fru), stachyose (gal(α1 → 6)gal(α1 → 6)glc(α1 → 2)fru), verbascose (gal(α1 → 6)gal(α1 → 6)gal(α1 → 6)glc(α1 → 2)fru) occur in legumes and brassicaceae. Beano® = α-galactosidase from Aspergillus niger breaks these sugars down and prevents the sometimes unpleasant and socially embarrassing consequences of eating these otherwise healthy foods.

23.1.3. Lactose intolerance In mammals lactase activity is highest in infants and declines in later life. If they ingest milk later the undigested lactose is fermented by endgut bacteria, resulting in flatulence, abdominal pain, and diarrhea (this is the reason why one should not give milk to cats). In some human populations with shepherding tradition lactase activity remains high throughout life (persistent lactase): Europeans, Arab bedouins, Tuareg, Tutsi and Fulani.In these populations the regulatory sequences responsible for switching off lactase production in adolescence are mutated. However, in other populations (e.g. Asians) [lactase] drops to 5– 10 % of the original level (non-persistent lactase). After ingestion of large amounts of milk or milk products, people with non-persistent lactase show signs of lactose intolerance. They can use only those milk products where lactose has been destroyed by fermentation (e.g. kefir). We distinguish Congenital hypolactasia lactase not present at birth and throughout life (inherited deficiency) primary hypolactasia decrease of lactase activity after weaning, in humans at age 5 a or so. secondary hypolactasia after damage to intestinal mucosa

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Biochemistry and Genetics

Figure 23.5.: Zymogen activation of pancreatic enzymes. Trypsinogen after release into the small intestine is activated by enteropeptidase. The resulting trypsin can then activate all other zymogens, including trypsinogen.

Figure 23.6.: 3D-structure of chymotrypsinogen and chymotrypsin.

446

Intermediary metabolism

23.2

23.1.4. Zymogens Digestive proteases and phospholipases are secreted as inactive precursors called zymogens. This mechanism protects the synthesizing cells from self-digestion. Glycosidases and lipases are not secreted as zymogens. Pepsin: Derived from pepsinogen. A 44-amino acid peptide is cleaved from the amino end of pepsinogen. This cleavage is catalyzed by pepsin or occurs by auto-activation at low pH. The cleaved peptide acts as a pepsin inhibitor at neutral pH. Pancreatic zymogens: Trypsinogen is activated to trypsin by the duodenal enzyme enteropeptidase. Trypsin then activates all pancreatic zymogens including trypsinogen. As an additional safety device, the pancreas contains a trypsin inhibitor. This inhibitor is a small polypeptide which binds trypsin with very high affinity. In acute pancreatitis, the pancreatic zymogens are activated within the pancreas and the pancreas digests itself and the surrounding adipose tissue, amylase and lipase are found in serum.

23.2. Intermediary metabolism Types of metabolic processes Anabolic reactions are biosynthetic reactions in which a complex product is made. They require metabolic energy. Catabolic reactions are degradative reactions. They produce useful energy as ATP. ATP is required for: • Biosynthetic reactions • Active transport across membranes • Cell motility and muscle contraction

Energy value of major dietary nutrients. Carbohydrates = 16.7 kJ/g (4.0 kcal/g) Fat = 38.9 kJ/g (9.3 kcal/g) Protein = 16.7 kJ/g (4.0 kcal/g)

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23.2

Biochemistry and Genetics

On a typical American diet, about 40 % of the energy comes from carbohydrates, 40 % from fat and 20 % from protein. In many indigenous people carbohydrate supply up to 80 % of the daily energy intake. Recommended are about 60 % from carbohydrates, 10 % each from saturated, mono-unsaturated and poly-unsaturated fatty acids, 10 % from proteins. Energy requirement per day Basal metabolic rate (BMR): 84–105 kJ/kg (20–25 kcal/kg) Post-prandial thermogenesis: 419–1675 kJ/d (100–400kcal/day) Muscular activity: variable Internal organs have high metabolic rates. The brain alone accounts for 25–30 % of the BMR. Muscle tissue (40–50 % of body weight) consumes 30 % of the BMR. Since ♂ have on average more muscle than ♀ (who have more adipose tissue), they tend to have a higher BMR. Metabolic regulation Metabolic processes are organized in pathways in which each reaction is catalyzed by a separate enzyme. Metabolic pathways are regulated: Biosynthetic pathways are regulated by feedback inhibition. Catabolic pathways are feedback-inhibited by ATP. All metabolic pathways can be regulated by hormones. Usually, the enzyme catalyzing the first irreversible reaction of the pathway (its committed step) is regulated. This implies that the enzyme catalyzing the committed step is present at lower activity than the other enzymes of the pathway. Mechanisms: • Enzyme synthesis may be regulated. • Allosteric regulation of key enzymes. • Phosphorylation of key enzymes. Compartmentalization of metabolism: Cytoplasm storage of fat and glycogen, glycolysis, pentosephosphate-pathway Mitochondria Krebs-cycle, oxidative phosphorylation, β-oxidation. Part of gluconeogenesis, heme and aa metabolism ER and Golgi protein, cholesterol and lipid synthesis, biotransformation. Glc6-phosphatase Lysosomes degradation of endocytosed and cellular macromolecules

448

Carbohydrate metabolism

23.4

Peroxisomes Oxidases that produce H2 O2 (FAD-dependent): very long-chain FA degradation, bile acid, cholesterol and plasmalogen synthesis. In plants: Glyoxylate shunt.

23.3. Carbohydrate metabolism Fate of glucose: Most dietary carbohydrate is absorbed as glucose. Most of the fructose and galactose is converted to glucose or glycogen in the liver. Metabolic fates in the cell: • Initially, glucose becomes phosphorylated to glucose-6-phosphate. This product can no longer leave the cell. • The major catabolic pathway for glucose is glycolysis. It takes place in the cytoplasm. Converts glucose to pyruvate and produces a small amount of ATP. • Excess glucose is used for glycogen synthesis. • There are several minor pathways of carbohydrate metabolism that make specialized products.

Acetyl-CoA mitochondrial oxidation Under aerobic conditions, pyruvate goes into the mitochondria where it is converted to acetyl-CoA. This metabolite is formed not only from carbohydrates, but also from fatty acids and amino acids. Acetyl-CoA feeds into the TCA cycle. The TCA cycle is therefore the final common pathway for the oxidation of all major nutrients. The catabolic pathways produce reduced coenzymes (NADH + H+ and FADH2 ). These are re-oxidized by the respiratory chain in the inner mitochondrial membrane. This strongly exergonic process drives ATP synthesis by oxidative phosphorylation.

Anaerobic glycolysis The conversion of glucose to lactic acid is our only possibility to produce energy under anaerobic conditions, but lactic acid tends to accumulate and lower the tissue pH. Importance: • Sudden high energy demand, for example in contracting muscle. • Absence of mitochondria, for example in erythrocytes. • Absence of oxygen, for example during ischemia.

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23.4

Biochemistry and Genetics

Figure 23.7.: Digestion of dietary fat (triglycerides) in the small intestine 1

H2C

O

O HC O O H2C 3 O 2

O 1- palmitoyl -2-oleoyl -3- linoleoyl-glycerol example for a fat (triacylglycerol) H2O pancreatic lipase

HO fatty acid

O H2C

O

O HC O O H2C OH

1,2-diacylglycerol H2O

pancreatic lipase

HO O

H2C

OH

HC O O OH

H2C

fatty acid

2-monoacylglycerol

Blood glucose Brain and erythrocytes require glucose at all times, therefore a constant blood glucose level of 70–100 mg/dL has to be maintained. The liver produces glucose by glycogen breakdown during short-term fasting and by gluconeogenesis (mostly from amino acids) during long-term fasting. Regulation is mostly hormonal.

23.4. Fat metabolism Triglyceride transport Fatty acids, mono- and diacylglycerol are absorbed by enterocytes by passive diffusion across microvillar membrane, together with fat-soluble vitamins. Inside the enterocytes FA are bound to intestinal FA binding protein to protect the cell membrane from its detergent-like effects. After re-synthesis of triacylglycerols they are packed in lipoproteins (chylomicrons) and released into lymphatics.

450

23.4

Fat metabolism

Figure 23.8.: Pancreatic lipase (cyan→purple) and colipase (yellow→green) PDB-code 1lpa. The enzyme becomes active only when contact with the lipid-water-interface removes the lid (deep blue helix) from the substrate site. The enzyme is a Ser-dependent hydrolase (similar to Ser-dependent proteases!)

Table 23.1.: Types of lipoproteins. density (g/ml) diameter (nm) mass (MDa) protein (%) phospholipid (%) triacylglycerol (%) free cholesterol (%) cholesteryl ester (%) apolipoproteins

Chylomicrons

VLDL

IDL

LDL

HDL

< 0.95 75–1200 400 1.5–2.5 7–9 84–89 1–3 3–5 A-I,A-II, B-48, C-1, C-II, C-III, E

< 1.006 30–80 10–80 5–10 15–20 50–65 5–10 10–15 B-100, C-I, C-II, C-III, E

1.006–1.019 25–35 5–10 15–20 22 22 8 30 B-100, C-I, C-II, C-III, E

1.019–1.063 18–25 23 20–25 15–20 7–10 7–10 35–40 B-100

1.063–1.210 5–12 0.175–0.360 40–55 20–35 3–5 3–4 12 A-1, A-II, C-I, C-II, C-III, D, E

Lipoproteins are non-covalent aggregates (“micelles”) of lipid and protein. The lipids have to be transported in this form because they are not sufficiently water soluble. Lipoproteins have a hydrophobic core (triacylglycerol, cholesteryl ester) and an amphiphilic coat (apolipoprotein, phospholipid, cholesterol). Their density is determined by content of fat (0.91 g/ml), cholesterol (1.067 g/ml), phospholipid (≈1.13 g/ml) and protein (≈1.5 g/ml). The coat is 2.0 nm thick in all cases: smaller diameter → higher density. Various lipoproteins have different function: Chylomicrons transport food lipids and cholesterol from intestine through the body VLDL, IDL, LDL transport cholesterol and lipid from the liver to other tissues (“bad cholesterol”) HDL transport cholesterol and lipids from other tissue to liver, scavenger of cholesterol (“good cholesterol”), liver converts cholesterol to bile acids

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Biochemistry and Genetics

Fate of liver synthesized cholesterol: • endogenous cholesterol and lipids packed in VLDL • lipid broken down by lipoprotein lipase like dietary lipids • VLDL remnants (IDL) converted to LDL LDL metabolism supply tissues with cholesterol: receptor mediated endocytosis 1. • Drug The body has to dispose of xenobiotics: non-nutritive and potentially • lysosome releases such fatty acids and amino acids into cytoplasm harmful substances as food-bourne and air-borne pollutants, drugs, and pyrolysis products in barbecue and cigarette smoke. Water-soluble substances • cholesterol + cholesteryl esters go to ER or droplets can be excreted, but lipid-soluble substances have to be converted into watersoluble products before they can be excreted in urine or bile. Drug metabolism is most active in the conversion liver but also occurs in other such as the and lungs. Carbohydrate-to-fat After a meal, excesstissues, dietary carbohydrate protein Hepatic drug metabolism proceeds in 2 stages. can be converted to fatty acids and triglyceride via acetyl-CoA, most are transported from Stage 1 metabolism oxidizes the drug through the microsomal the liver to other tissues as VLDL (very-low density lipoprotein). But fatty acids cannot cytochrome P-450 enzymes (mixed-function oxidase) be converted to glucose! Cyt-P-450 R-OH + H20 + NADP+ R-H + 02 + NADPH + H+

are more than 100 different molecular forms of cytochrome P-450. 23.5. There Metabolism of amino acids and protein

They have broad and overlapping substrate specificities and can theefore oxidize a wide variety of drugs, environmental pollutants, and even endogenous steroid Amino acids and protein typically 100 g, and 30–40 g are hormones. Thisprotein system Dietary is induced by intake many isdrugs, for about example barbiturates. the minimum requirement. The levels of free amino acids are low in the body, but there are This results in tolerance formation, often cross-tolerance when one drug induces 10–15 kg of for protein. Daily proteinofsynthesis enzymes the metabolism another (and drug.degradation) is about 300 g. This consumes 5 % of the basal metabolic rate. About half the amino reactions acids can be synthesized Stage 2 metabolism consists of of conjugation with glucuronicin the body, others needsulfate, to be taken up withhydrophilic food (essential amino acids). acid,the glutamine, or other groups. The water-soluble conjugation products are no longer biologically active and can be excreted in urine or bile. Catabolism of amino acids Amino acids are catabolized mostly in the liver. The nitrogen is converted to the excretory product urea (daily production: 30–35 g from 100 g protein). The nitrogen-free products formed from the amino acids are either oxidized to CO2 + H2 O or used for biosynthetic reactions, including gluconeogenesis. 2. Alcohol metabolism 23.6. Alcohol Metabolism Alcohol is derived from microbial fermentation: Alcohol is derived from microbial fermentation: ATP ADP, Pi + NADH CO2 NAD 1/2 Glucose

Pyruvate

NADH

Acetaldehyde

NAD+ Ethanol

Some ethanol is formed by intestinal bacteria and metabolized as a firstpass effect in the liver. Alcohol is absorbed from the intestine, also the stomach. It is diffusible 452 and therfore readily distributed throughout the body. 2% are excreted by lungs and kidneys, the rest is metabolized in the liver and to a lesser extent the stomach. The zinc-containing cytoplasmic enzyme alcohol dehydrogenase (ADH) is normally rate-limiting, as is the availability of NAD+. Alcohol oxidation is a zero-order reaction down to serum levels of about 40 mg/dL. 10 g are

23.6

Alcohol Metabolism

Some ethanol is formed by intestinal bacteria and metabolized as a first-pass effect in the liver. Alcohol is absorbed from the intestine, also the stomach. It is diffusible and therefore readily distributed throughout the body. 2 % are excreted by lungs and kidneys, the rest is metabolized in the liver and to a lesser extent the stomach. The zinc-containing cytoplasmic enzyme alcohol dehydrogenase (ADH) is normally rate-limiting, as is the availability of NAD+ . • Km for alcohol dehydrogenase is 1 mM (0.046 h → 0 order kinetics 0.15 h/h in most people • Km for aldehyde dehydrogenase 10 µM: no accumulation of ethanal metabolized each hour, and the blood level declines by 15 mg/100 mL/h. Pathway: Pathway: NAD+ Ethanol

NADH

NAD+

Acetaldehyde

NADH Acetic acid GTP, CoA-SH Acetyl-CoA

GMP, PPi

The cardiac value of alcohol is 7 kcal/g. Most of the acetic acid generated

in the liver iscontent transported to otheristissues for oxidation. The energy of alcohol 29 kJ/g. Most of the acetic acid generated in the liver is “Oriental flush” is a hypersensitivity transported to other tissues for oxidation. to alcohol, with facial flushing and

tachycardia after only one or two drinks. It is caused by a dominantly inherited deficiency of the mitochondrial aldehyde dehydrogenase, often in combination “Oriental flush” is a hypersensitivity to alcohol, with facial flushing and tachycardia after with a “superactive” ADH. This trait occurs in 40% of Chinese and Japanese. only oneDisulfiram or two drinks. It is caused by a inherited dominant negative used (tetramer!) deficiency (“Antabuse’) is an inhibitor of aldehyde dehydrogenase, to oftreat the alcoholics. mitochondrial aldehyde dehydrogenase (Glu487Lys), often in combination with a “suhigh (“Antabuse’) Alcohol metabolism [NADH] / [NAD+] ratio, peractive” ADH. This traitresults occursininan 40increased % of Chinese and Japanese. Disulfiram energy charge, and high mitochondrial acetyl-CoA. Pyruvate dehydrogenase is an inhibitor of aldehyde dehydrogenase, used to treat alcoholics. and TCA cycle are inhibited because of increased [NADH] / [NAD+] and [ATP] / [ADP]. Alcohol oxidation is not feedback-inhibited. Therefore alcohol is oxidized H2 H2 S in preference to S other nutrients. CH3 pyruvate and oxaloacetate are C H3C Gluconeogenesis inhibitedC because N C S S is C N+ leads to a high [lactate] / [pyruvate] depleted: the high [NADH] / [NAD ]Cratio 3 H3CandCa high [malate] / [oxaloacetate]CH ratio ratio (equilibrium of LDH and malate H 2 H 2 dehydrogenase!). Hypoglycemia and lactic acidosis can develop in alcohol intoxication.Disulfiram (Antabuse) Drug interactions:

+ ratio, high energy AlcoholAlcohol metabolism in an increased [NADH + enzymes H+ ] / [NAD inducesresults the synthesis of drug-metabolizing in the ]liver, charge, high their mitochondrial acetyl-CoA. Pyruvate dehydrogenase but alsoand inhibits activity acutely. Barbiturates, for example, work inand the TCA cycle are + ] and [ATP] / [ADP]. Alcohol oxiintocxicated but notofthe sober alcoholic. anesthesia! inhibited because increased [NADHImportant + H+ ] /in[NAD dation is not feedback-inhibited, the body wants to get rid of this toxin as soon as possible. VI. BLOOD CELLS Therefore alcohol is oxidized in preference to other nutrients.

Mature RBCs have no mitochondria. Glucose is metabolized by anaerobic glycolysis (ATP formation) and the pentose-phosphate pathway (NADPH) formation. Only 20g are consumed per day. Synthesis of 2, 3-bisphosphoglycerate (BPG):

254

453

23.7

Biochemistry and Genetics

Gluconeogenesis is inhibited because pyruvate and oxaloacetate are depleted: the high [NADH + H+ ] / [NAD+ ] ratio leads to a high [lactate] / [pyruvate] ratio and a high [malate] / [oxaloacetate] ratio (equilibrium of LDH and malate dehydrogenase!). Hypoglycemia and lactic acidosis can develop in alcohol intoxication. Other health effect of alcohol • in form of distilled spirits provides energy, but no minerals, vitamins, essential AA... • increases the fluidity of the PM → neurotoxicity • converted to triglycerides → lipidemia, fatty liver • Replacement of hepatocytes by connective tissue: cirrhosis • Mitochondrial damage, leaky inner membrane • Aldehydes modify and cross-link proteins

23.7. Intermediary metabolism Catabolic and anabolic processes in our body are closely intertwined, which is only possible by tight regulation. Regulation of pathways occurs by substrate concentration according the the HMM-law. The binding/dissociation equilibrium is diffusion controlled, that is, almost instantaneous. allosteric regulation (also very fast). Homo- and heterotropic effects.

enzyme modification works on an intermediate timescale (minutes). Phosphorylation/dephosphorylation is the best-studied example, but remember that there are many other reactions as well. enzyme synthesis/degradation is slow, on a timescale of hours to days. It is also expensive in metabolic energy. To make a good regulatory site, an enzyme has to meet several criteria: • the product of the enzyme should be precursor of a single pathway (no downstream branch-points). • the enzyme should be the first irreversible step of the pathway. Regulating reversible reactions is not necessary (they are not metabolically expensive). If the regulated enzyme were behind the first irreversible step of a pathway, intermediates would accumulate and potentially become toxic. • the activity of the enzyme must be low compared to all other enzymes of the pathway (it must form the bottleneck).

454

Digestion: Objectives in summary

23.9

Figure 23.9.: Regulation of metabolic pathways. Top: Feedback inhibition of an anabolic pathway. The substrate A is converted into the product Z via a number of intermediates. Once the concentration of Z is high enough, it shuts down its own production. Note that the regulation could not occur at the earlier reaction step, since that would also prevent the production of other products, to which B is precursor. Middle: Feedback inhibition of a catabolic pathway. Product of this pathway is ATP, which serves as feedback regulator. At the same time, ADP may stimulate the reaction (example: phosphofructokinase). Thus the rate of food breakdown depends on the energy requirements of a cell. Bottom: Feedforward stimulation. The presence of a substrate activates the pathway for its catabolism (example: Lac-operon). B

A

C

z

Feedback inhibition (anabolic)

C

z

Feedback inhibition (catabolic)

z

Feedforward stimulation

other products

A

B

ADP

A

B

C

ATP

23.8. Questions 1. How much fat can you lose per day on a 0 J diet (tap water and vitamin pills)? 2. Why is it so difficult to lose only fat and not also muscle on a strict weight reduction diet? 3. Glucose utilization by the brain is not insulin-dependent. What does this mean for glucose use by the brain in the fasting state?

23.9. Digestion: Objectives in summary 1. List the enzymes for the digestion of major nutrients including, starch, disaccharides, proteins, triglycerides, phospholipids and nucleic acids, and their site of action in the ali-

455

23.9

Biochemistry and Genetics

mentary tract. 2. Name those digestive enzymes that are subject to zymogen processing, and state the mechanisms of zymogen activation. 3. Describe the role of mixed bile salt micelles for lipid absorption in the small intestine. 4. Predict the effects of total gastrectomy, pancreatic failure and biliary obstruction on the digestion of major nutrients. 5. Know why some people get flatulence after drinking too much milk. 6. Define the terms “anabolic”, and “catabolic”, and state the roles of cytoplasm, lysosomes, endoplasmic reticulum and mitochondria in intermediary metabolism. 7. Describe the principles of feedback inhibition and feedforward stimulation in anabolic and catabolic pathways. 8. Predict in general terms the consequences of genetic deficiencies, enzyme-inhibiting toxins and vitamin/coenzyme deficiencies on different types of metabolic pathways. 9. Elaborate the characteristic features of regulatory enzymes of metabolic pathways.

456

24. Heme, Purines and Pyrimidines 24.1. Heme Biosynthesis Heme is made in all cells according to their needs. About 250 mg/d total, 75–80 % of this in the bone marrow, 15 % in the liver. The uncolored porphyrinogens have −CH2− groups connecting the pyrrole rings, and the colored (and fluorescent) porphyrins have =CH− groups. The porphyrinogens can be oxidized non-enzymatically to the corresponding porphyrins by exposure to light and air. δ-aminolevulinate synthase is the rate-limiting enzyme. In most cells, transcription and transport of the enzyme (S1-isoform) into mitochondria are inhibited by heme and hematin (heme with Fe in the ferric form), which also act as allosteric inhibitors. In addition, ALA is allosterically inhibited by glucose. Erythroblasts of the bone marrow express the S2-isoform (encoded on the X-chromosome), which is up-regulated by EPO. The enzymatic activity is allosterically stimulated by iron.

24.1.1. Disorders of Heme Biosynthesis Porphyrias are diseases in which abnormal quantities of porphyrin or its precursors are excreted. They are caused by a partial deficiency of one of the biosynthetic enzymes other than ALA synthase, either in the liver or bone marrow: heme formation is reduced, and ALA-synthase is dis-inhibited. The accumulation of toxic intermediates causes attacks of abdominal pain, neurologic signs, and/or cutaneous photosensitivity. Treatment is possible by hematin infusion. Also dietary carbohydrate is effective because it represses ALAsynthase. Intermittent acute porphyria is a hepatic porphyria, autosomal dominant with deficiency of uroporphyrinogen I synthase. Porphobilinogen and δ-aminolevulinate appear in the urine. Symptoms do not appear before puberty. Drugs and steroid hormones which induce the synthesis of heme-containing hepatic enzymes (cytochromes P-450 enzymes) trigger attacks of abdominal pain, vomiting, neuropsychiatric, and cardiovascular abnormalities. Only a minority of individuals with the genetic trait ever have attacks.

457

O

H2 P C

O CH

+

N

O CH3

-

CH2

COOH CH2

+

C O

H2O

δ-ALA

CH2

+

Glycine

δ-ALA synthase

CH3

O

H2O H + N C COOH H2

H3N

NH3 C COO H2

H

HC

PLP (Vit. B6)

H2 P C N

+

COOH H

CO2 HC

+

H

N

Tyrosinemia I (Succinylacetone)

CH

3

N

H2O

lead-poisoning (replacement of catalytic Zn)

COOH

Fasting

LIVER

CH3 C CH2 H

+

2+

4 NH3

HOOC C C H2 H2 HOOC C H2

H3C HOOC C C H2 H2

HO C H2

H2 C

COOH

HN

CH2

COOHCH2 CH2

N H

H N

CH2

CH2

CH2

COOH

CH3

HN

CH

CH2

COOH

CH3

N H

H N

CH2 CH2

CH2

H C

CH

2 O2

H2C

COOH

CH2

COOHCH2

H2 C

HN B

CH2

CH2

H N

COOH

C

N H

A

CH2

D NH

C H

2

CH2

COOH

N H

CH3

NH

H N

CH2

CH2

HN

CH3

C COOH H2

C C COOH H2 H2

CH3

C C COOH H2 H2

CH2

H2 C

COOH

CH2

C

H2C

porphyria cutanea tarda

4 CO2

Uroporphyrinogen decarboxylase

Uroporphyrinogen-III (UROGEN)

HOOC C C H2 H2

HOOC C H2

congenital erythropoetic porphyria

H2O2

H2O

Uroporphyrinogen-III synthase Hydroxymethylbilane

C COOH H2 C C COOH H2 H2

2

CO2

H3C

HOOC C C H2 H2

CH2

COOH

CH2

but: -increased demand -> precursors leave cell - δALA and PBG: neurologic symptoms (neuro-visceral + neuro-psychiatric): GABA antagonist! - Uro- + Koproporphyrinogens: skin lesions by photosensitization, fragility, itch

Porphyrias: - dominant inheritance: 50% activity of enzyme -> reduced [heme] -> activation of δ-ALA synthase -> normally sufficient heme synthesis

H2 Coproporphyrinogen-III (COPROGEN, hydrophobic)

hereditary coproporhyria

2

Coproporphyrinogen oxidase

CH3 C CH2 H

CH

C CH2 H

3

Protoporphyrinogen oxidase also spontaneous

CH3

N

CH

CH2

COOH

CH3

N H

H N

CH2

COOH

CH2

H2 C

CH2

NH

C H2

H2C NH

C H2 Protoporphyrinogen IX (PROTOGEN, even more hydrophobic!)

3 O2

HC

C H

N

3 H2O2

porphyria variegata

HC 3

Protoporphyrin IX (PROTO, conjugated system, colored, red fluorescence)

Ferrochelatase HOOC C C H2 H2

protoporphyria lead-poisoning

Fe

acute intermittend porphyria (madness of King George III)

11q24, splice variants in erythroblast (42 kDa) and all cells (44 kDa)

PBG-desaminase

CH2

COOHCH2

N H

CH2

NH3

CH

H C

Glucose

CH

2

CH

2+

N

CH3

2H

Mitochondrial matrix

Cytosol

Porphobilinogen (PBG)

Fe N

CH2 CH

H2C

2 H2O

δ-ALA dehydratase

+

HC

C H

N

PBGS-porphyria (rare)

COOH CH2 CH2 H C O + N CH2 O CH3

HOOC C C H H

2

H3C

transcription transport into mito allosteric inhibition

H2 P C

3p21: all cells (S1-isoform) Xp11-21: erythroblasts (S2 isoform)

COOH CH2 CH2

O CH 3

H C O + N C COOH H H2O

CH2 CH2 C

+

+

H

N

HC

S CoA

H2 P C

+

Fe

2+

CoA SH

Succinyl-CoA

EPO

Erythroblast transcription translation

Sideroblastic anemia: - iron-loaded mitos form ring around nucleus - defect in δ-ALA synthase 2 - lowered affinity for pyridoxin

2

Heme

2

COOH

458

Biochemistry and Genetics

24.1.1

Figure 24.1.: Heme biosynthesis.

Heme Degradation

24.2.1

Porphyria cutanea tarda is the most common porphyria. Uroporphyrinogen decarboxylase is deficient, but symptoms occur only in patients with iron overload and liver damage. Cutaneous photosensitivity is the most prominent symptom. Photosensitivity occurs in all those porphyrias in which porphyrins (rather than porphyrin precursors, as in intermittent acute porphyria) accumulate. δ-Aminolevulinate dehydratase and ferrochelatase are inhibited in lead poisoning: δ-aminolevulinate is increased in the urine and protoporphyrin IX in RBCs.

Laboratory tests δ-Aminolevulinate and porphobilinogen can be measured in the urine with colorimetric tests. Coproporphyrinogen and uroporphyrinogen are spontaneously oxidized to the corresponding porphyrins if the urine is left standing in air. The can be extracted from the urine, then identified spectrophotometrically or -fluorimetrically.

24.2. Heme Degradation About 85 % of the heme in the body is present in hemoglobin. 6 g of hemoglobin are degraded per day, most of this in the spleen. The heme becomes first biliverdin, then bilirubin. Heme oxygenase (heme → biliverdin) is only CO-forming enzyme in the body. Bilirubin is transported to the liver in non-covalent binding to albumin. Liver uptake requires a facilitated diffusion type carrier of high capacity, the organic anion transport protein (OATP). The liver conjugates bilirubin to water-soluble bilirubin diglucuronide which is actively secreted into the bile canaliculi by the canalicular multiple organic anion transporter (cMOAT), also known as multidrug resistance related protein 2 (Mrp2), an ABC-type transport ATPase. This step is rate-limiting for hepatic bilirubin metabolism. Both the UDP-glucuronyl transferases and bilirubin secretion into bile are stimulated by various drugs, including phenobarbital. If there are problems with excretion of bilirubin diglucuronide into bile, Mrp3 will export the conjugate into the blood stream. Bacteria in the terminal ileum and the colon remove the glucuronate residues, convert bilirubin to colorless urobilinogens, and the urobilinogens to colored urobilins (stercobilin etc.) which are responsible for the brown color of the stools. Some urobilinogen is absorbed and undergoes entero-hepatic circulation. Trace amounts are also excreted in the urine. The normal serum bilirubin concentration is < 1 mg/dL. Most of this is unconjugated.

459

H3C

2

COOH C C H H 2

HC

D N C H

CH3

N

A

CH2 CH H C

N B

CH

CH2

O

CH3

O

3 [O]

O O HO O

OH

O

CH3HN

N

P

CO

P

HC P

3

HC

C H

N

CH3

N

Fe N

V

2+

2

O

N

O 3

OH

O

CH V

O

OH

CH

OH

O

P CH3 Verdoglobin (green)

2 UDP

UDP-GT1-A1 (perivenous liver cells)

2 [H]

Globin 3+

Fe

O UDP

E

Urobilinogen

bacterial enzymes

2 [H]

bacterial glucuronidases

Crigler-Najjar-Syndrom (< 10% remaining act.)

Morbus Meulengracht (icterus intermittens juvenilis, ~30% remaining activity, 8% of population)

E

CH3

Glucuronic acid

E

CH3

CH2

O

newborn jaundice

N H

HN CH3

CH3

C H

O

Stercobilin (golden)

H2 C

N H H3C

CH3

N H

H2C HO

C H 2

O

V

Note: about 1% of blood hemoglobin complexed with CO

C CH2 H

E

H H2C N

O

Hemoglobin (red)

CH3

2+

Fe N

C CH2 CH 2

O

O

CH3

O

H2C

OH

OH

O

N H H3C

COOH

V

O Bilirubin diglucuronide (direct bilirubin)

HO

Polymers (dark color of stool)

E O

HN CH3 CH3 H2 P CH2 C N N H N C H H HC 3 P Urobilin (orange-yellow) excreted in urine -> yellow color

CH3

A N H

N

D

P

C

H3C

P

O

P C H2

CH2

N H

O HN B

CH3 E

Urobilinogen

N H

HN CH3

O

bacterial enzymes

V

CH3

O

H N

V

CH3 P

H N

P Lumirubin

N

no hydrogen bridges between propionate and NH-groups of ring A and B -> more water soluble -> excretion by the kidney

N H CH3

low water solubility -> transported by albumin if albumin capacity exceeded: - jaundice (deposition in skin + sklera, from Fr. jaune = yellow) - kernicterus (deposition in brain)

λ = 460 nm

V

CH3

Biliverdin reductase

Spleen macrophages

Biliverdine (blue-green)

H3C

Heme oxygenase (CYP450)

V O

+

+

NADP

P

H N

HN CH3 CH 3 P H N N H N C H H2 H3C P Bilirubin (orange-red) (indirect bilirubin)

8 [H]

H2 C N H H3C

CH3

O

NADPH + H

V

O

re-uptake into blood, transport to kidney

O

CH3

460

Biochemistry and Genetics

24.2.1

Figure 24.2.: Heme degradation.

24.2.1

Hyperbilirubinemia and Jaundice

Figure 24.3.: Transport and metabolism of bilirubin in the hepatocyte. ATP

ATP

Bile

Mrp3

ER

ABCC3 ADP + P

Mrp2

Blood

cMOAT ABCC2 ADP + P Ligandin

OATP Albumin

24.2.1. Hyperbilirubinemia and Jaundice

Conjugated or unconjugated bilirubin or both are elevated in various diseases. Both kinds of bilirubin deposit in sclera and skin, causing jaundice (=icterus). However: • Only unconjugated bilirubin can enter the brain and cause kernicterus. This occurs in newborns when the binding capacity of serum albumin (25 mg/dL at normal albumin concentration) is exceeded. Kernicterus can be fatal, and survivors may be left with a motor disorder and mental deficiency. • Only conjugate bilirubin can be excreted by the kidney. In choluric jaundice, the urine is dark yellow-brown (i.e., urine dark and stool pale). Serum bilirubin is measured by the van den Bergh method. A colored product is formed. Direct (reacting) bilirubin is bilirubin-diglucuronide which can be determined directly in aqueous solution. Indirect (reaching) bilirubin is unconjugated bilirubin. It reacts poorly in water because of its tight binding to albumin. It is measured after the addition of an organic solvent like methanol or ethanol.

461

24.2.1

Biochemistry and Genetics

Types of hyperbilirubinemia Mixed hyperbilirubinemia 1. Hemolytic diseases can cause mild hyperbilirubinemia (usually < 4 mg/dL). 2. Physiological jaundice of the newborn is the most common form of jaundice. It is caused by immaturity of the liver and is usually self-limited. If treatment is required, you can use: • Phototherapy, which destroys excess bilirubin in the skin. • Phenobarbital, to induce the metabolizing enzymes in the liver. • Exchange transfusion if serum bilirubin approaches 20–25 mg/dL. 3. Conjugated disease of the newborn is most often caused by rhesus incompatibility. It may require exchange transfusion immediately after birth or even before birth. Conjugated hyperbilirubinemia Elevated conjugated with almost normal unconjugated bilirubin indicates biliary obstruction (cholestasis), either intrahepatic (bile canaliculi blocked) or posthepatic (hepatic or common bile duct blocked). Obstructive jaundice can be caused by gallstones, malignancies, or severe acute liver diseases. Chronic biliary obstruction can cause liver damage (biliary cirrhosis)! Unconjugated hyperbilirubinemia is seen in many liver diseases including infections (hepatitis, yellow fever) and toxins (carbon tetrachloride, acetaminophen, α-amanitin in mushroom poisoning). Urobilinogen can be measured in urine, blood, and stools. In cholestatic jaundice, urinary and fecal urobilinogen disappear. The stools of these patients are clay-colored while the urine is brown. Urinary urobilinogen is high in hemolytic jaundice and in nonspecific liver damage.

Inherited defects of bilirubin metabolism Crigler-Najjar syndrome type I is a severe congenital form of unconjugated hyperbilirubinemia (20 mg/dL), usually fatal in infants. It is caused by a complete deficiency of UDP-glucuronyl transferase. Crigler-Najjar syndrome type II is much milder, with a lot of bilirubin monoglucuronide in the bile. Caused by a partial deficiency of the bilirubin-conjugated enzyme.

462

24.3.1

Purine Biosynthesis

Dubin-Johnson syndrome is a defect in the secretion of conjugated bilirubin and other amphophilic substances conjugated to glutathione or glucuronic acid (defect in the ABCC2 transporter (canalicular multiple organic anion transporter (cMOAT)), with conjugated hyperbilirubinemia. Course of the disease is relatively mild, treatment is not usually required.

24.3. Purines 24.3.1. Purine Biosynthesis

Most of our purines come from endogenous synthesis: IMP, AMP, GMP ADP, ATP, GDP, GTP

Purine biosynthesis O from pentose phosphate pathway

O P O CH2 O O

ATP

OH

O

O

AMP

O P O CH2 O O

O

GART

O

P

O

O

P

PRPP

Gln

Glu

O

O

NH2

ATP

O

NH2

ADP + Pi

O P O CH2 O O

O

NH

Formyl-THF

Rib P OH OH

OH OH

Rib-5-P

Phosphoribosyl pyrophosphate (PRPP)

Gln-PRPP amino transferase

Gly

OH OH

PPi

THF

Phosphoribosyl amine

O -

COO O COO

-

N H

Asp

C

O

C

CO2

N

H2N

N

H2N

N

H2N

N

HN

Rib P

Rib P

Rib P

H N

H2O

N

N

Glu

H N

Gln

C O H O

NH Rib P

AIRC

C O H

NH Rib P

ADP+Pi

ATP

Adenylosuccinate lyase Fumarate

COO

O H2N

C

H2N

N

Formyl-THF

THF H2N

N

O

Rib P

O

O C N H

IMPS

H2O

N

HN

N

C

GTP

N

-

H2 COO C HC NH

GDP+Pi HN

N

N

Rib P

Rib P

Inosine monophosphate (IMP)

Note: GTP is used for the synthesis of AMP ATP is used for the synthesis of GMP

NH2

C

N

N

N

HN

Rib P

Asp

N N Rib P

Furmarate

Adenylosuccinate synthetase

C N

AMP

Adenylosuccinatete lyase

H2O GMP synthetase O HN H2N

AMP+PPi

C

N

N

N Rib P

GMP

IMP dehydrogenase

HN O Glu

Gln

NAD

+

GTP

AMP

O

ATP

C

N

N H

N Rib P

Xanthosine monophosphate (XMP)

NADH + H

+

AMP desaminase

GMP reductase

GMP

NH4

+

GDP XMP NH4

ATP

GTP

+

The amidotransferase catalyzes the committed step in purine synthesis. Regulation is by feedback inhibition:

463

24.5

Biochemistry and Genetics

Ribose 5-P

PRPP

Phosphoribosylamine

IMP

Inhibited by AMP

Inhibited by purine nucleotides

AMP

Inhibited by GMP GMP

VI. PURINE DEGRADATION AND SALVAGE REACTIONS. Purines are degraded to uric acid: Adenine nucleotides

Guanine nucleotides

24.3.2. Purine Degradation Pi

Pi Inosine

Guanosine Ribose 1- P

Ribose 1-P

Hypoxanthine Guanine Purines are degraded to uric acid (see fig. 24.4). Uric acid is the only important product of purine degradation in humans. Xanthine Oxidase Xanthine

NH3

Xanthine Oxidase

O2

24.4. Pyrimidine Metabolism O H N

HN O

O N

Uric Acid

N

H H Pyrimidines are synthesized form carbamoyl phosphate and aspartate. Carbamoyl phosphate is made by a cytoplasmic enzyme distinct from the mitochondrial carbamoyl phosphate synthetase of the urea cycle. Ribose 5-phosphate (from PRPP) is added after the Uric acid is the only important product of purine degradation in humans. synthesis of the pyrimidine ring (see fig. 24.5).

Salvage reactions recycle the bases by reacting them with PRPP:

The cytosolic carbamoylAdenine-phosphoribosyl phosphate synthetase is transferase feedback-inhibited by CTP. Adenine + PRPP

AMP + PPi Hypoxanthine-guanine Hypoxanthine + PRPP Phosphoribosyl transferase IMP + PPi Guanine + PRPP (HGPRT) GMP +that PPi convert orotic Familial orotic aciduria is caused by a deficiency of one of the enzymes

acid to the uridine nucleotides. With growth retardation, megaloblastic anemia, and a reactions reduce acid formation, and they the auxotrophs need crystallineThese sediment of orotic aciduric in the urine. The patients areminimize pyrimidine who for de novo synthesis. respond to large doses of orally-administered uridine. A secondary form of orotic aciduria (but without dependence on exogenous pyrimidines) also occurs in those urea cycle enzyme VII. PYRIMIDINE METABOLISM. deficiencies in which carbamoyl phosphate accumulates. Carbamoyl phosphate leaking out of the mitochondria bypasses the rate-limiting step of pyrimidine biosynthesis. 233

464

24.5 Pyrimidine Metabolism

Figure 24.4.: Purine degradation N

N

Rib

N

N

+

OH

N

OH

N

N

Inosine

N

Rib

N

Pi Rib P

Rib

P

Purine nucleoside phosphorylase

Pi

N

N

OH

OH

N

OH

N

N

N

N

H2O2

O2+ H2O

O2+ H2O

N

N

Hypoxanthine

N

Purine nucleoside H2N N H2N N N N phosphorylase Rib Guanine Guanosine Guanine PNP deficiency (#613179): T-cell deficient, desaminase B-cells normal, neurologic problems (pyramidal signs, spaticity) dGTP accumulation -> low ribonucleotide reductase -> low [dNTP]

NH4

Adenosine desaminase

H2O

Purine degradation

2

NH

N Adenosine

ADA deficiency (#102700): severe combined immunodeficiency (SCID) NK-, T- and B-cells cannot develop -> "bubble baby" [dATP] increased 100 times -> ribonucleotide reductase downregulated -> low [dNTP] -> no cell division additional problems with S-adenosylhomocysteine hydrolase -> no DNA methylation -> apoptosis Treatment: PEGylated bovine ADA (i.m.), bone marrow transplant

Allopurinol

N

NH4

H2O

HO

+

OH

N

Rib

N Xanthine Xanthine oxidase

N N

OH

P

N

Mo, 4 FeS, FAD

H2O2

HO

Pi

N Oxypurinol

N

suicide inactivation of xanthine oxidase by Allopurinol

HO

N

OH

N

N

N

+

N

O

N

N

N

HO

O

N

H N

O

elevated uric acid -> gout

O

N

lactam-form

HN

pKa = 5.75

urate

ROS

CO2

O

in animals (except primates, reptils, birds) further enzymatic degradation to - allantoin (mammals) -allantoate (bony fish) - urea (amphibians, selachians) - ammonia (marine invertebrates)

Uric acid

HN

N

Rib

N

OH

H

HO

Xanthosine

N

Purine nucleoside phosphorylase

N

OH

O2+ H2O

N

H2O2

HO

lactim-form

N

O NH2

spontaneous

O

Allantoin

465

Growth factors

UTP

PRPP

N C

O

+

Gln

H2N

Carbamoylphophate synthetase II

+

(cytosolic) O

CH2

H C O C

O

+ CO2

C O

2 ATP

2 ADP + Pi + Glu

CAD

O

O

C

C N

C

O

CH

Dihydroorotase

CO2

+

orotidylate decarboxylase

NADPH + H

NAD

+

ribonucleotide reductase

CH dRib

N

CH

UMP

P Rib

C P

dUMP

H2O C

O

2

CH

H N H C O C C

C

C

O

N

O

C

CH C P Rib Orotidylate

PPi

O

O

+

C

H N

O

C

CH

C

+

O

NADH + H

NAD

C HN

O

Orotate

UMP synthase

defective in hereditary orotate aciduria (interferes with growth and development, hypochromic anemia) Treatment: oral uridine

O

HN

Orotate phospho- PRPP ribosyl transferase

(inner membrane of mitos)

Dihydroorotate dehydrogenase

O Dihydroorotate

HN

O

orotate aciduria in some urea cycle defects: mitochondrial carbamoylphosphate spills into cytosol and is converted to orotate -> relieve of nitrogen stress

Pi

O P O

2 ATP

ATP

Asp-transcarbamoylase

CTP

2 ADP

UMP kinase NDP kinase

O

C

C

O

N

CH

H N H C O O C C H2N CH2 C O O

C

H2O

O

HN

Methylene-THF

Thymidylate synthase

DHF

HN

O

CH

O Asp

ATP Gln

O

HN

N-carbamoylaspartate

Pi ADP Glu

Cytidylate synthetase

CH

Carbamoyl phosphate

H3N

MAP kinases

CH

CH

NH2 C N

UTP

P P P Rib

Pyrimidine biosynthesis

O P P P Rib CTP

C

O

CH dRib

N

CH3

lack of folate: dUTP build into DNA instead of dTTP -> strand breaks from DNA repair enzymes -> cancer Folate antagonists: Methotrexate, sulfonamides

P

C

HN O

dTMP

466

Biochemistry and Genetics

24.5

Figure 24.5.

24.6

Deoxyribonucleotides

24.5. Salvage pathways

Salvage reactions recycle the bases by reacting them with PRPP: O

O

O O

O P O CH2 O O

P

HN

O O

O

P

O

O

N H Uracil

O

PPi

OH OH PRPP

NH2

O

O

N

N

N Adenine

N

N H

H2N

PPi

or

N

Rib Thymidine

N

N Rib P

IMP

ADP O CH3

HN

N

Rib P AMP

O

Salvage pathways

O

O

N H2N

UMP

PPi

O

N

N

Thymidine kinase

vide infra

N

O

CH3

HN

ATP

NH2 N

O

Rib P

N H

Guanine

Hypoxanthin/guanine phosphoribosyl transferase (HGPRT)

N

N N

O HN

N

N N

N H Hypoxanthine

Adenin phosphoribosyl transferase (APRT)

Uracil phosphoribosyl transferase (UPRT)

N

N N

N

N

Rib P TNP Thymidine kinase activates Aciclovir to the active form, which terminates the chain in viral DNA synthesis

Rib P GMP

These reactions reduce uric acid formation, and they minimize the need for the energyexpensive de novo synthesis.

24.6. Deoxyribonucleotides

Deoxyribonucleotides are required only for DNA synthesis and repair. They are present at concentrations well below those of the corresponding ribonucleotides. Reaction:

467

24.7

Biochemistry and Genetics

H2 P P O C O

Base

H2 P P O C O

OH OH

Base

OH H

nucleotide diphosphate (NDP)

deoxynucleotide diphosphate (dNDP) H2O

activity regulation:

substrate specificity:

dATP

ATP/dATP: reduction of UDP, CDP

ATP

dTTP: reduction of GDP dGTP: reduction of ADP SH

ribonucleotide reductase

SH

either:

S

or:

SH

S Glutaredoxin

S

ribonucleotide reductase

Glutaredoxin

SH

S

SH

S Thioredoxin

Thioredoxin

SH

S

Glutaredoxin reductase 2 GSH

GSSG

FADH2

Thioredoxin reductase

FAD

Glutathione reductase NADP

+

NADPH + H

+

NADP

+

NADPH + H

+

24.7. Anti-neoplastic and anti-bacterial drugs acting on nucleotide metabolism The dihydrofolate (DHF) formed in the thymidylate synthase reaction has to be reduced back to tetrahydrofolate: dihydrofolate DHF + NADPH + H+ GGGGGGGGGGGGGGGGGGA THF + NADP+ reductase Some anti-cancer drugs inhibit nucleotide synthesis: Fluorouracil is converted to fluorodeoxy-UMP, an irreversible inhibitor of thymidylate synthase. It is also incorporated in RNA in place of uracil. Methotrexate (amethopterin) is a folate antagonist and inhibits dihydrofolate reductase. Azaserine is a glutamine antagonist which inhibits the use of glutamine for purine synthesis, the IMP → GMP and the UTP → CTP reaction.

468

Hyperuricemia and Gout

24.8

Bacteria, unlike humans, can synthesize their own folic acid, and rely on this capability to support their rapid division. Sulfonamides inhibit this reaction competitively with paminobenzoic acid. Today they are usually given together with a dihydrofolate reductase inhibitor like trimethoprim, so that any folate produced or obtained from the host can not be converted into the physiologically active form. Virus also require nucleotides for their reproduction by the cell, nucleotide analogues may be used to interfere.

24.8. Hyperuricemia and Gout 300–600 mg uric acid are produced per day, and 80 % of this is excreted in the urine. The normal plasma concentration is 2–7 mg/dL. It is about 1 mg/dL lower in females than in males and increases with age. Clinical problems result from the low water solubility of uric acid. Uric acid is a weak acid with pKa of 5.8. The acid form (at low pH) is less soluble than the salt form (at neutral or alkaline pH), but also the sodium salt is poorly soluble when [Na+ ] is high. Solubility • in urine: 15 mg/dL at pH 5, 150 mg/dL at pH 7 • in serum: 7 mg/dL Sustained hyperuricemia (serum urate > 7 mg/dL) causes sodium urate deposits in soft tissues (tophi), and in joints where they cause the inflammatory response of gouty arthritis. About 0.5 % of adult male and 0.1 % of females are affected. Secondary gout is caused by an underlying disease. Most important are diseases with extensive tissue destruction, including chronic hemolytic anemias, myeloproliferative disorders, psoriasis, and malignancies. Radiation or chemotherapy of malignant tumors can cause rampant hyperuricemia. Primary (idiopathic) gout is not caused by another disease. There is either uric acid overproduction or impaired renal excretion. Patients with more than 600 mg uric acid in the 24-hour urine (15–25 % of patients) are overproducers. Some overproducers have increased activity of PRPP synthetase or decreased activity of HGPRT. A complete deficiency of HGPRT causes Lesch-Nyhan syndrome, an X-linked recessive disease with mental retardation, spasticity, self-mutilation and rampant hyperuricemia. Treatment of gout: Colchicine and non-steroidal anti-inflammatory drugs are used to treat the acute attack.

469

HN

O F

2 ATP

HN O

O

N

F

Rib P

thymidylate synthase

FdUMP

dTMP

7,8-DHF

Trimethoprim Sulfonamides

+

Gln-Analoga

Glutamine

+

NH3 H2 H2N C CH COO C C H2 O

SH N

Purin biosynthesis

N

C

OH

Rib

N

Cl

N

S

+

N

N

N

CH3

NH3 O H N C CH COO C C H2

Cyt

OH

N

O NH2

Ribavirin inhibits IMP-DH after phosphorylation

Rib

N

N + + NH N 3 H2 C CH COO C O Azaserine

HC

O

N

Precursor of 6-Mercaptopurine

Azathioprin

N

N

Acivicin

O2N

H2 HO C C

OH OH

O N H

Hydroxyurea (radical scavenger)

H2N

Cyclopentyl cytosine gets phosphorylated and inhibits CTP synthetase

Ribonucleotide reductase

gets phosphorylated and then inhibits OMP decarboxylase

Deazauridine

O

Pyrimidine biosynthesis

N N Folate synthesis (bacteria only) 6-Mercaptopurine inhibits Gln-PRPP aminotransferase and adenylosuccinate metabolism

Methotrexate Aminopterin

NADPH + H

+

Cyt

NADP

araCTP

OH

HO

H2 P P P O C O

DHF-reductase

2 ADP

THF

Folate cycle

dUMP

O N H Fluorouracil also inhibits mRNA processing

Cyt

N5,N10-methylene THF

C1-body from aa breakdown

H2 HO C O HO OH Cytosine arabinoside

inhibits DNA polymerase competitively with dCTP

470

Biochemistry and Genetics

24.8

Figure 24.6.

Objectives in Summary

24.9

Probenecid is a uricosuric drug which inhibits the renal tubular reabsorption of uric acid. Allopurinol is an inhibitor of xanthine oxidase. Less uric acid is formed, and the patient excretes a mix of hypoxanthine, xanthine, and uric acid. A high-purine diet (meat) can worsen gout, as the nucleotides contained are turned into uric acid directly by the enterocytes. Alcohol causes acidemia, which lowers uric acid solubility, worsening gout. This is the explanation for the gout attacks sensitive patients suffer in the morning after a night of overindulgence. Cave: diabetic ketoacidosis! Uric acid stones account for 5–10 % of all kidney stones. Treatment: increased fluid intake, alkalinization of the urine, allopurinol.

24.9. Objectives in Summary • Summarize the principles of heme synthesis and degradation. Explain the pathomechanism of acute and chronic porphyrias, sideroblastic anemia and hyperbilirubinemia. Sketch how these conditions can be diagnosed and treated. • Summarize the principles of purine synthesis and degradation. Explain the pathomechanism of PNP-deficiency, SCID and hyperuricemias. Sketch how these conditions can be diagnosed and treated. • Summarize the principles of pyrimidine synthesis and degradation. Explain the pathomechanism of orotate aciduria and folate deficiency. Sketch how these conditions can be diagnosed and treated. • Describe how nucleosides and bases are salvaged and state the significance of these pathways. • Explain the mechanism of anti-neoplastic and anti-infectious drugs that act on nucleotide metabolism.

471

25. Nutrition During the Life Cycle 25.1. Nutrition in Pregnancy and Lactation Good eating habits are essential in pregnancy • To ensure that the mother maintains optimal nutritional status, which will be reflected in the way she feels and behaves and her ability to cope with normal duties. • To permit normal growth and development of the fetus in utero. • To ensure the delivery of a baby of normal birth weight, >2500 g. Low birth weight is associated with ill health and infections in the neonatal period. Stages of pregnancy: 1. The periconceptional phase, up to 4 weeks after conception. Good nutrition during this time period is important for normal implantation, normal cell divisions in the embryo, and development of the placenta. 2. The period of organogenesis, 4-12 weeks after conception. This period requires a balanced nutrition to permit normal fetal development. It is also the period of greatest sensitivity to teratogens. 3. The period of rapid growth, 12 weeks - delivery. This period requires a substantial increase in bulk nutrients (protein, calcium, iron) to support fetal growth. Commonly encountered problems: • Poor eating habits up to the time of conception resulting in poor nutrient stores: iron, vitamin A, folic acid • Nutritionally unbalanced slimming diets at or before conception. • The mother may have had repeated pregnancies. • The use of drugs and alcohol, especially during the phase of organogenesis, exposes the fetus to teratogenic risks (fetal alcohol syndrome, cocaine-baby). Excessive alcohol intake also affects the absorption, metabolism and excretion of many nutrients, particularly zinc, magnesium, copper and iron. There are important physiological changes during pregnancy:

473

25.1

Biochemistry and Genetics

• In the mother: – Metabolic rate increases. – Enlargement and growth of uterine walls – Development of breast tissue in preparation for breast-feeding – Development of the placenta – Laying down of fat stores for lactation – Increase in maternal blood volume by 20–50 %. This requires extra synthesis of hemoglobin and albumin. – Pregnancy hormones are produced by the corpus luteum (maternal origin) and the placenta (fetal origin). – Blood loss must be anticipated during delivery. • In the baby: – The fetus requires nutrients for rapid growth. – The baby has to build up iron stores for 3 months. The physiological processes demand increases in protein, zinc, calcium, iron, iodine, folate, and vitamins C, A, and B6. Folic acid is extremely important because deficiency in early pregnancy increases the risk of neural tube defects (anencephaly and spina bifida). Pregnancy demands no special food, but a balanced and adequate supply of all essential nutrients. US Recommended Daily Allowances (RDAs) . Nutrient Not pregnant Pregnant Energy (kJ) 9200 1st trimester 10500 2nd + 3rd trimester 11700 Protein (g) 50 60 Calcium (mg) 800 1200 Iron (mg) 15 30 Folate (µg) 180 400 Iodine use iodized salt Vitamin C (mg) 60 95 Vitamin A (µg) 800 1300 Vitamin D (µg) 8 10

474

Nutrition in Pregnancy and Lactation

25.1

All B vitamins should be increased. For vitamin A, both excess and deficiency have to be avoided because excess vitamin A is teratogenic. 3. Weight gain during pregnancy: Women who enter pregnancy 10 % or more below or 20 % or more above standard weight for height have a greater risk of poor pregnancy outcome. During the first trimester the mother should expect to gain 900–1800 g. During the last six months a weight gain of 500 g per week is acceptable. As a rule of thumb, weight gain should be between 11 and 12.5 kg for the entire pregnancy. Underweight women and teenagers may need to gain more. Reducing diets are not recommended during pregnancy. Women who do not gain weight often have underweight babies. However, large increases in caloric intake are not required because placental hormones facilitate fat breakdown in the mother’s adipose tissue. Tissue/Fluid Infant at birth Placenta Increase in blood volume Uterus and muscles Increase in breast tissue Amniotic fluid Mother’s fat stores

Weight gain (kg) 3.5 0.5 1.8 1.1 1.4 0.9 1.8–3.6

Calcium: Dairy products are the most convenient source of calcium. Zinc: Nuts, shellfish and meat supply ample amounts but a diet too high in fiber will impair absorption. Iron: There are substantial amounts of iron in whole wheat bread, oats and other whole wheat cereals. A good supply of vitamin C is required for efficient iron absorption. Because heme iron is more readily absorbed than other forms of iron, animal foods should be included in the diet. Minor complaints during pregnancy Relaxation of the bladder and intestinal walls is caused by pregnancy hormones. This can lead to urinary tract infections and constipation. At least 8 glasses of fluid should be taken to counteract urine stasis, whole wheat cereals, vegetables and fruit should be taken to prevent constipation. Most mothers experience lack of appetite, early morning sickness, nausea, and aversion to certain types of food (‘pregnancy sickness’). This is typical between weeks 4 to 12, at the time of organogenesis. Pregnancy sickness is a normal defense mechanism that causes the mother to avoid potentially toxic foods at a time when the fetus is most sensitive to teratogens.

475

25.2.1

Biochemistry and Genetics

Smoking, alcohol, and caffeine-containing beverages should be discouraged. All pregnant women are advised to take iron supplements daily, especially as anemia is a problem in some prenatal clinics in the US (10 %). Fruit juice should be taken with iron supplements to achieve maximum absorption.

25.2. Breastfeeding Human milk is adapted to the precise needs of the young. With the exception of water and lactose, human milk and cow’s milk are dissimilar in almost all respects. Colostrum, the thin yellowish liquid that precedes mature milk, is often present before the end of pregnancy but is secreted mainly during the first 5 post-partum days. It contains less fat and lactose than mature milk and more sodium, chloride, and zinc.

25.2.1. Composition of Human Milk The protein content is about 11 g/L, one third of that in cow’s milk. Casein is particularly low, resulting in soft curd and easy digestibility. α, β-lactoglobulin in cow’s milk is a common food allergen in infancy. Milk allergy causes eczema, diarrhea and asthma. Human milk does not contain β-lactoglobulin but harmless α-lactoglobulin. Human milk is higher in taurine and cystine and lower in tyrosine and phenylalanine than cow’s milk. This composition is important for the pre-term infant whose liver is inefficient in converting methionine to cysteine and in metabolizing phenylalanine and tyrosine. Human milk fat contains 14 % polyunsaturated fatty acids (linoleic acid). The cholesterol content is some 10 to 20 times higher than in cow’s milk. Fat-soluble vitamins are present, but vitamin K is low. All other vitamins are present in adequate amounts. Human milk is rich in lactose (7 %). The sodium concentration of human milk is relatively low. This is important because of the limited capacity of the newborn kidney to deal with a heavy load of solute. The calcium is absorbed better than the calcium in cow’s milk. Iron, copper, manganese, zinc, magnesium and iodine are present only in small amounts. The iron content of human milk is 0.2–0.3 mg/L. Close to 50 % of this is absorbed. The low iron content is advantageous because iron is an essential nutrient for pathogenic bacteria. Iron is made unavailable to bacteria by tight binding to lactoferrin (in milk) and transferrin (in the blood). Excess iron would disrupt this protective effect. Full-term babies don’t need much iron because they are born with a 3-months supply of iron. Only premature babies require iron supplements. The absorption of zinc from human milk is more effective than that from cow’s milk because of its association with a different zinc-binding protein. Human milk contains antibodies of the IgA type. Colostrum is particularly rich in IgA. Enteric antigen-stimulated plasma cells in the mother migrate to the breast tissue where

476

Dietary recommendations for lactating mothers

25.2.2

they secrete antibodies or are themselves secreted into the milk where antibodies are produced. Breast milk is known to prevent gastroenteritis in infants. Human milk (but not cow’s milk) also has a high content of lysozyme. Leucocytes are present in human colostrum. 90 % are macrophages, and 10 % are IgAproducing lymphocytes. Sterilizing breast milk will completely destroy the cellular as well as many of the protein components of breast milk and eliminate their antibacterial effects. Post-partum amenorrhea is favored by breastfeeding, poor nutritional state, and physical illness. Mothers in good nutritional state, however, resume cycling 2–6 months after birth in spite of continued breast-feeding. In non-contracepting human populations (as in other animal species), the birth interval is determined by the duration of post-partum amenorrhea.

25.2.2. Dietary recommendations for lactating mothers Item Energy (kJ) Protein (g) Iron (mg) Fluids

Non-lactating 9200 50 15

Lactating 11300 65 15 Extra fluids

The amount and composition of milk is affected by the mother’s nutrition: in malnourished mothers, lactose remains constant but the pattern of fatty acids charges. The fatty acid composition of the milk reflects that of the mother’s diet. Concentrations of water soluble vitamins reflect the mother’s dietary intake. Most drugs ingested by a lactating mother will appear in her milk. Moderate alcohol consumption by lactating women is considered acceptable. Breast-feeding depends on the functioning of reflexes in both the baby and the mother. The ‘rooting’, ‘suckling’ and ‘swallowing’ reflexes are present in newborns. Very small premature babies may have weak reflexes. The mother has 2 important reflexes: The prolactin reflex involves afferent nerve impulses from the nipple and areola to the hypothalamus and the stimulation of prolactin release by lactation. The milk ejection reflex involves afferent impulses to the hypothalamus. The hypothalamus induces the release of oxytocin, a hormone which contracts the myoepithelial cells in the mammary gland. Emotional tension and stress inhibit this reflex, and the main cause of lactation failure is thought to be inhibition of this reflex. The suckling of the infant and consequent release of oxytocin decrease maternal uterine bleeding and hasten the return of the uterus to its normal size.

477

25.3

Biochemistry and Genetics

The baby should be fed ‘on demand’ rather than by the clock. Because of its small stomach capacity and rapid growth, the baby requires frequent feeding during the first few weeks. Breast milk is the only food required by the baby during the first four months.

25.3. Feeding the Weaning Age Group The weaning period is a transitional period, between the time the baby is fed entirely with milk and the time she is feeding entirely on the adult diet. This is the period between 4 months and 2.5 a of age. • Eating habits introduced during childhood are likely to continue until adulthood, therefore good eating habits should be introduced early. • From the age of about 4 months, the baby is able to swallow semisolid foods. There is a need for increased nutrients at this time, particularly energy and iron. • Weaning should be gradual. Abrupt removal of breast milk is totally unacceptable. Force-feeding and bullying is to be strongly discouraged as the baby will develop a hatred for food, associating mealtime with punishment. • New foods should be introduced one at a time to reduce the risk of allergic reactions. • The consistency of the meals is very important. Sieved vegetables, soft fruits, or root crops with some gravy should be offered, followed by mixtures of two types of food (sieved beans and root crop) and later still mixtures of three or four types of food (e.g., chicken, bean, potato, vegetables). Sieved foods should be replaced by mashed foods by eight months and chopped foods by one year. • Because of the small capacity of baby’s stomach, and her high nutrient needs, feedings must be small and frequent, and nutrient-dense food must be given. • Exploring and experimenting are normal and desirable behavior patterns at that age. A child’s impulses, if constantly denied, can turn to shame and self-doubt. A child wanting to feed herself, making a mess at mealtime, or refusing to eat, behaves normally for her age. Strategies must be used to deal with these behaviors, but no attempts should be made to stifle them. • There are advantages and disadvantages of commercial foods and family pot. Provided that the family pot is nutritionally balanced and free from pathogens, it has the same benefits as the commercial varieties. The following is a guide to the ratio of the different foods to be used during preparation. Animal Food 1 Tablespoon Vegetables 1 Tablespoon

478

Feeding the School Child

25.5

Starch 4 Tablespoons Legumes 2 Tablespoons Mothers may choose between feeding meals from the ‘family-pot’ or using one of the commercially prepared varieties. Special care must be taken to ensure that baby’s share is removed before adding seasoning and salt and that bones and pits are removed. Popcorn and nuts are forbidden and sweet foods are not to be encouraged. The practice of allowing babies to continue bottle-feeding in bed leads to dental caries and buck-teeth.

25.4. Feeding the School Child The aims are: • To ensure that dietary intakes include a balance of macro- and micronutrients which will enable the child to achieve optimal physical and mental development. • To establish good dietary habits that are carried over into adulthood, for the prevention of diseases later in life. These diseases include coronary heart disease, hypertension, osteoporosis, diabetes, and cancer. The recommended distribution of calories is the same as for adults: Fat 30 %, protein 10– 15 %, carbohydrate 55–60 %. Growth is affected by the diet, but anthropometric measurements must be interpreted with caution. Children have growth spurts that take place at different ages in different children, most obviously at puberty. On average, the highest growth rate for boys is during the fifteenth year, but it is much earlier for girls. Cognitive development is affected by nutrition. Better nutrition has been credited with most or all of the Flynn effect: the secular increase to IQ that has been observed over the past century. The Flynn effect is evident from preschool age to adulthood. Commonly encountered problems in school children: • Iron deficiency anemia is caused by poor diet in times of rapid growth. • Obesity is caused by poor eating habits and lack of exercise. • Dental caries are caused by the frequent consumption of sucrose. Streptococcus mutans, the most important caries bacterium, polymerizes sucrose into a dextran that glues the bacteria to the tooth enamel. The sugars in fruits and vegetables and the lactose in milk and dairy products are less cariogenic than sucrose.

479

25.6

Biochemistry and Genetics

25.5. Feeding Adolescents On average, the growth spurt for females begins at age 10–11 a and reaches a peak at 12 a. In males, it typically begins at age 12–13 a and peaks at 14 a. In females fat becomes a larger percentage of total body weight and in males the lean body mass, muscle and bone, becomes much greater. There are also hormonal changes that produce physically and, hopefully, mentally mature adults within 2–3 years. There is tremendous variation in teenagers’ rates and patterns of growth. Energy Needs A rapidly growing 15 a old boy may need about 17 700 kJ/d. Girls of the same age have stopped growing and a girl of 15 a may need only 8400 kJ/d, if she is not to become obese. Iron An ample intake of iron-rich foods should be encouraged in teenagers. Iron is important for a rapidly growing body, especially a female body that needs extra iron for menstruation. The iron loss during each menstrual period is between 10 and 25 mg. Iron needs pose a problem with teenagers because • Traditional snack foods are low in iron. • Some teenagers may be vegetarians. • Some may adopt weird diets or may be bordering on anorexia nervosa. Calcium The requirement for calcium reaches a peak during these years. Some teenagers reject milk as “child’s drink’ - choosing sodas instead. Nutritious types of snack should be encouraged. Emphasis should be placed on vitamin C in citrus fruits and vegetables, deemphasizing soft drinks which only provide empty calories. French fries and greasy burgers are high in salt and fat. Dairy products should be encouraged. Nuts and beans should be encouraged for their vitamin B and iron contribution to the diet.

25.6. Nutrition in the Elderly Features of the aging process that are important for nutrition of the elderly: • Poverty • Ill-fitting dentures • Lack of appetite due to reduction in taste buds

480

Nutrition in the Elderly

25.6

• Decreased metabolic rate due to total reduction of body cells • Constipation, indigestion, chronic diseases Most important: The elderly need love. Many elderly take drugs on a regular basis. Common drug interactions: Anticonvulsants affect vitamin D metabolism and folate absorption. Biguanides (oral anti-diabetics) reduce B12 and folate status. Tetracycline reduces leucocyte levels of ascorbic acid and increases its urinary excretion. Aspirin treatment over time depletes the tissue stores of ascorbic acid and induces chronic G.I. bleeding leading to iron-deficiency anemia. Thiazide diuretics Iron deficiency anemia in the elderly can be caused by: • Reduced gastric acid secretion, which impairs iron absorption. • Heavy reliance on tea and toast. The tannins in tea prevent iron absorption. • Chronic blood loss: ulcers, hemorrhoids. • Prolonged use of antacids which interfere with iron absorption. The elderly require less energy due to their decreased metabolic rate. However too low an energy intake may lead to a decreased intake of other nutrients. Inadequate protein intake can lead to low hemoglobin levels (anemia), with apathy and fatigue. Encourage low-calorie sources of high-quality protein: low-fat dairy products, fish, liver, lean minced meat. Protein should supply 12 % of the total energy intake. Fat should be limited to keep calories down and to lessen the risk of atherosclerosis, cancer, and arthritis. Fat should represent 20 % of total caloric intake, mainly poly- and monounsaturated which will lead to a reduction in blood cholesterol. Fiber-rich foods help prevent constipation, and they keep the blood cholesterol down. Whole-grain cereals, root crops and fruits contribute to the energy content of the diet while also providing significant quantities of vitamins. Mineral deficiencies e.g. depletion of K+ , has been associated with muscle weakness and mental confusion. Depletion of zinc can lead to decreased taste acuity, anorexia and delayed wound healing. An adequate intake of calcium is necessary to prevent osteoporosis. Milk and milk products and fish with soft bones are good sources. Milk can be incorporated into the diet in porridge, yogurt and ice cream.

481

25.7

Biochemistry and Genetics

Fluids are important. Some elderly people have a fading sense of thirst. Some fear fluids to avoid incontinence and may become dehydrated in the process. Dehydration can lead to confusion, headaches, and irritability. Physical activity is very important for the well-being of the elderly. It prevents muscular atrophy and the development of osteoporosis, and it also increases the blood flow to brain and limbs. Fruits and vegetables should be emphasized as sources of vitamins and minerals. Overcooking leads to destruction of vitamins B and C, and vitamin E is usually destroyed in some canned processed foods, therefore advice should be given on how to buy, cook and store vegetables, with emphasis on food spoilage, which may lead to diarrhea. Sunshine is important to provide vitamin D for the bones.

25.7. Objectives in Summary 1. Articulate the dietary guidelines and recommendations for Americans. 2. Define the nature and types of dietary protein and its contributions to the nutritional content of the diet. 3. Name the coenzyme forms of water-soluble vitamins niacin, riboflavin, thiamine, B6, pantothenic acid and folic acid. 4. Describe the deficiency syndromes for niacin, thiamine, ascorbic acid, folic acid, vitamin B12 and vitamins A, D and K. 5. Name those vitamins that function as antioxidants. 6. Outline the metabolism and inter-organ transport of vitamins A and D. 7. Explain the reasons for bone demineralization in rickets. 8. Describe the approximate distribution of iron in the body and the functions of iron containing proteins ferritin, hemosiderin and transferring. 9. List typical situations in which iron deficiency anemia is encountered. 10. Describe the origin, clinical presentation and treatment of iron overload. 11. State the importance of hemoglobin concentration, total iron binding capacity, iron saturation of transferring and serum ferritin as laboratory test for the evaluation of the iron status. 12. Identify dietary sources of heme iron and non-heme iron, and explain factors which inhibit and enhance iron absorption.

482

Objectives in Summary

25.7

13. Explain the term “protein sparing action of carbohydrates”. 14. Define the metabolic rate and the factors which influence the metabolic rate. 15. Discuss energy requirements in relation to physical activity. 16. Articulate the basic principles, advantages and disadvantages of a vegetarian and vegan diet 17. Describe the chemical composition, caloric value and food sources of fats. 18. Define osteomalacia, osteoporosis and outline their causes and prevention. 19. Take a diet history and analyze its content. 20. List the nutritional needs in pregnancy, lactation, childhood, adolescence, and the elderly. 21. Identify measures to enhance calcium, iodine, protein, vitamins A and C, folate and iron contents of diets during pregnancy. 22. Explain the importance of breastfeeding during infancy, and the precautions necessary for successful bottle feeding. 23. Discuss the treatment of the diarrheal child. 24. Define obesity and describe its harmful effects and its prevention. 25. Explain the causes, prevention and treatment of kwashiorkor, marasmus and failure to thrive in infancy. 26. Describe the types, consequences and dietary treatments of hypercholesterolemia. 27. Prepare diets to meet the caloric and other nutritional needs of patients suffering from hypertension, diabetes type I and II, coronary heart disease, obesity, anorexia nervosa and kwashiorkor. 28. Discuss the role of desirable lifestyles in control of chronic diseases and define the importance of nutritional education to combat chronic diseases. 29. List the recommended guidelines for fat intake as directed by the American Health Association. 30. Describe the role of diet in the management of renal failure, nephrotic syndrome, hepatic coma, liver cirrhosis, hepatitis, cholecystitis, peptic ulcer, burns, post surgery, trauma and malabsorption syndromes. 31. List the principles of enteral and parenteral nutrition. Define the major types of food sensitivity: Celiac disease, egg, peanut and soya allergy, and lactose intolerance.

483

26. Cell Cycle Control and Cancer 26.1. Cell Cycle Control Dividing cells proceed through the cell cycle, while resting cells are in G0. G0 is a diploid state like G1, but it is physiologically different: Many genes that are required for cell cycle progression are expressed in G1 but not G0. Conversely, many genes connected with the terminal differentiated phenotype of a cell is expressed in G0 but not in G1. Apoptosis is “programmed” cell death. It occurs during normal embryonic development, in regenerating epithelia, in response to hormones, and in response to severe damage, especially DNA damage. The induction of specific proteases is a key event in apoptosis. Two mechanisms are important for cell cycle control: • Proteins that are required for specific stages of the cell cycle are produced only at that time. Cyclin proteins orchestrate these changes. Many enzymes of DNA synthesis (DNA polymerase α, the polymerase δ subunit PCNA, thymidylate kinase) are transcriptionally induced shortly before S phase and degraded shortly after. • Other proteins are present throughout the cell cycle but are regulated by phosphorylation. Some structural proteins become phosphorylated at the start of mitosis: chromosomal scaffold proteins assemble in response to phosphorylation, and the lamins of the nuclear lamina disassemble. Also many transcription factors and other regulatory proteins become phosphorylated and dephosphorylated at different points in the cell cycle. There are two main checkpoints at which the cell makes an all-or -none decision: • The G1 checkpoint is in late G1. The cell commits itself to DNA replication. • The G2 checkpoint is in late G2. The cell commits itself to mitosis. Note that the processes of DNA replication and mitosis cannot be interrupted once initiated. (Interrupting the cell cycle in metaphase with the drugs colchicine or colcemid will eventually lead the cell to die.)

485

26.3

Biochemistry and Genetics

26.2. Normal Cells in Culture Most cells can be grown outside the body in suitable nutrient media. Properties of cultured cells: Mitogen dependence Mitogens are growth factors that are necessary for cell division. Purified growth factors (EGF, PDGF) can be used, or serum. Serum (but not fresh plasma) contains PDGF, released from activated platelets during blood clotting. Also nonphysiological mitogens (phytohemagglutinin for leucocytes) can be used. Anchorage dependence All cells, except leukocytes, require a solid support for growth in culture. Contact inhibition Cells are normally inhibited by neighboring cells. Therefore cultured cells grow only until a continuous monolayer has formed. Mortality Cultured cells divide many times in culture (50–100 times in the case of fibroblasts), then go into senescence and die. These limitations on cell growth apply to normal cells, not cancer cells.

26.3. Cyclins and the Retinoblastoma Protein The cell cycle is regulated by the concerted activities of nuclear protein kinases and protein phosphatases. Cyclins are the regulatory subunits of nuclear protein kinases. Important cyclins: Cyclin D: Present throughout the cell cycle. It is induced by mitogens. Activation of this cyclin provides an important link to extracellular hormones. MAP kinase and PI3K/Akt signal transduction pathways lead to cyclin D1 activation, and cell cycle progression. Cyclin E: A “G1-cyclin”. It brings the cell through the G1 checkpoint. Cyclin A: Most abundant during S phase and early G2. Cyclin B: The classical “G2-cyclin”. It brings the cell through the G2 checkpoint and is required for the phosphorylation of chromosomal scaffold proteins and lamins. It is degraded suddenly during mitosis. Control of the G1 checkpoint: • Entry into S phase requires the transcription of genes for DNA polymerases and other proteins of DNA replication. Transcription of these genes requires the dimeric transcription factor E2F/DP.

486

p53 and the Damage Response

26.5

• E2F/DP is bound to the promoters of the regulated genes at all times, but during early and mid-G1 its transcriptional activator domain is masked by the tightly bound retinoblastoma protein (pRb). • pRb is a phosphoprotein. Only the hypophosphorylated form suppresses E2F/DP. • At the G1 checkpoint, pRb becomes phosphorylated by complexes of cyclin D and cyclin E with cyclin-dependent protein kinases (Cdks). As a result, pRb falls off the transcription factor and the E2F/DP regulated genes are expressed. pRb is the “guardian of the G1 checkpoint”.

26.4. p53 and the Damage Response Cells have two possibilities to respond to DNA damage: 1. Passage through the G1 checkpoint is blocked, and repair enzymes are induced. The cell must not replicate its DNA until all the damage is repaired! 2. The cell undergoes apoptosis. This prevents survival of aberrant, irreversibly damaged cells. The choice between these two alternatives depends on the cell type and the kind and severity of the damage. Not only DNA damage but also other kinds of stress (heat stress, or inhibition of transcription or translation) can trigger these responses. Sequence of events leading to apoptosis: • DNA damage, oxidative stress, hypoxia and telomere erosion induce the synthesis and inhibits the degradation of the p53 protein. ATM kinase also phosphorylates p53 causing it to accumulate in the nucleus. • p53 is a transcription factor which binds, in an oligomeric form, to the regulatory sites of some genes. It stimulates the expression of genes for DNA repair and cell cycle arrest, and/or for apoptosis. • One of the p53-induced genes codes for the Cdk-inhibitor p21 (= waf-1). p21 causes cell cycle arrest. The mediators of apoptosis are not well known.

487

26.5

Biochemistry and Genetics

26.5. Growth Control by External Stimuli In order to establish and maintain the normal histology of the body, every cell has to respond to a multitude of external stimuli. External stimuli can affect: • The growth rate of the cell. • The rate of cell division. Growth rate and mitotic rate are usually regulated in parallel. • Cell motility. Most cells can creep, and they do so most obviously during embryonic development and wound healing. • Cell shape. Both motility and shape changes require regulation of the cytoskeleton. External stimuli include: Cell-cell contact Primary cells in culture stop dividing when they contact other cells. There are many different cell adhesion proteins. They are integral membrane proteins. Most of them make homotypic interactions with the same adhesion proteins on neighboring cells. They interact with the cytoskeleton through peripheral membrane proteins. Cellcell contact triggers cytoskeletal remodeling and signaling cascades into the nucleus that mediate contact inhibition. Cell-matrix contact Primary cells in culture have to contact a polystyrene or glass surface in order to grow. In vivo, cells make contact with the extracellular matrix. Cell-matrix interactions are mediated by receptors of the integrin type. Along with peripheral membrane proteins, attached cytoskeletal proteins, and signaling proteins (protein kinases, G-proteins), they form the focal adhesions. Cell-matrix contact affects cell shape and motility and is required for normal growth (anchorage dependence!). Growth factors Cells respond to soluble growth factors that stimulate or (less commonly) inhibit cell growth and proliferation. Most growth factors act locally in the tissue where they are formed, but some circulate as hormones. Also “ordinary” hormones and their second messengers can affect cell growth. The Phospholipase C-IP3-calcium system is mitogenic for many cells. cAMP inhibits the proliferation of white blood cells and fibroblasts but stimulates many endocrine cells.

488

26.6

Mitogenic Signaling

Growth factor Nerve Growth Factor (NGF)

Tissue of origin Sympathetically innervated tissues

Target tissue Sympathetic ganglia

Effect + Growth

Insulin-like Growth Factor-1 (IGF-1)

Liver

Many cells

+ Differentiation - Mitosis + Growth

Platelet-derived Platelets Many cells Growth Factor (PDGF) Epidermal Many cells Many cells Growth Factor (EGF) Fibroblast Many cells Many cells Growth Factor (FGF) Erythropoietin Kidney Bone marrow Transforming Many cells Many cells Growth Factor (TGF) Note: IGF-1 is also produced locally in many different tissues.

+ Mitosis + Mitosis + Mitosis + Mitosis + Differentiation - Mitosis

26.6. Mitogenic Signaling Mitogens trigger phosphorylation cascades that regulate the phosphorylation states of nuclear transcription factors and other regulators of gene expression. Most growth factor receptors have an intracellular protein tyrosine kinase domain. The activated receptors aggregate and phosphorylate each other. The autophosphorylated receptors bind cytoplasmic signaling proteins, thereby recruiting them to the plasma membrane, activating them allosterically, and/or tyrosine-phosphorylating them. This triggers several signaling pathways, including: Activation of phospholipase C (PLC) PLC has several isoenzymes. PLC-β is stimulated by hormone-activated G-proteins. PLC-γ becomes tyrosine-phosphorylated and thereby activated by growth factor receptors. The diacylglycerol formed by PLC activates protein kinase C (PKC). Activated protein kinase C stimulates NF-κB activity.

489

26.7

Biochemistry and Genetics

Activation of the MAP kinase pathway. 1. Through adapter proteins, the autophosphorylated growth factor receptor activates the Ras protein, a protein similar to G-protein GTP binding proteins. Unlike the hormone-regulated G-proteins, Ras has only one subunit, but it also cycles between an inactive GDP-bound form and an active GTP-bound form. 2. Ras-GTP activates the Ser/Thr protein kinase Raf. [Ras-GTP also activates PI3-Kinase which activates a protein kinase called Akt (Also known as Protein Kinase B). Akt activation inactivates inhibition of Cyclin D1 (Cell growth is stimulated).] 3. Raf phosphorylates and activates protein kinases of the MEK (= MAP kinase/Erk kinase) type. 4. The MEKs activate proteins of the MAP kinase family by phosphorylation on closely-spaced Thr and Tyr side-chains. 5. The MAP kinases enter the nucleus and phosphorylate nuclear proteins on Ser and Thr side-chains. They phosphorylate cytoplasmic target proteins as well, and they stimulate ribosomal protein synthesis non-selectively. The MAP kinase pathway is also stimulated by insulin in many cells. Receptors for cytokines, including interferons, interleukins, erythropoietin, prolactin, growth hormone and leptin, recruit a Tyr-specific protein kinase (Janus kinase) which activates a transcription factor (signal transducer and activator of transcription (STAT)).

26.7. Principles of Malignant Transformation Neoplasia (“new growth”) is the abnormal proliferation of a cell population that is derived from a normal somatic cell. Benign neoplastic conditions are self-limiting, but malignant ones (“cancer”) are deadly. Cancers are classified according to their cell of origin: sarcomas are derived from connective tissue cells and carcinomas from epithelial cells. Cancers originate from a normal somatic cell by malignant transformation, usually through somatic mutations. Therefore all mutagens are also carcinogens. The cells in a tumor are derived from a single aberrant somatic cell: cancers are monoclonal in origin. The malignant phenotype is heritable at the cellular level. Properties of cancer cells: Mitogen-independent growth Lack of contact inhibition De-differentiation Cancer cells are poorly differentiated, much like embryonic cells.

490

Cellular Oncogenes

26.8

Disordered growth Cancer growth is chaotic, without any respect for anatomical boundaries. Genomic instability Many cancer cells have chromosomal aberrations and/or a mutator phenotype. Immortality given nutritional requirements. High mitotic rate The number of mitoses in cytological specimens is important for the prognosis of cancer. Metastasis Cells break loose from the tumor, are carried away by lymph or blood, and establish secondary growths. Cancer cells can be cultured easily. They need no mitogens, there is no anchorage dependence or contact inhibition, and the cultures can be propagated indefinitely. With rare exceptions, more than one mutation is required to make a cell malignant. Tumors can vary in their degree of malignancy even if they are derived from the same cell type. This is because each tumor has its own personal combination of cancer-inducing mutations. Malignant tumors can arise from benign lesions, and low-grade malignancies can spontaneously transform into more malignant varieties. This is called tumor progression. It is caused by the appearance of new mutations in an already abnormal cell population. The more malignant mutants will always outgrow their less malignant neighbors. It’s evolution in the fast track! Oncogenes are genes in tumor cells whose expression promotes the malignant state. Oncogenes are derived by activating somatic mutations from normal cellular proto-oncogenes. Mutational activation leads either to structural alterations of the gene product, or its overproduction. Rarely, an oncogene is introduced by a virus. Tumor suppressor genes are normal cellular genes that inhibit cell proliferation or invasiveness. Their inactivation can make the cell malignant. Oncogenic activation is effective even if only one copy of a cellular proto-oncogene becomes mutationally activated, but mutations of tumor suppressor genes cause malignancy only if both copies of the gene are knocked out.

26.8. Cellular Oncogenes Oncogenes are derived genes from mutation of proto-oncogenes. Most oncogene products are involved in mitogenic signaling pathways. Thus the mutations are activating, and result in hyperstimulation of mitogenic signaling pathways.

491

26.9

Biochemistry and Genetics

• Growth factors are rare as oncogene products, but the retroviral sis (simian sarcoma) oncogene is a truncated version of PDGF. • Growth factor receptors are frequent oncogene products. Most of these oncogenes code for structurally abnormal growth factor receptors that are always switched on, even in the absence of the ligand. Examples: erb B, neu. Also overexpressed but structurally normal growth factor receptors are common in many cancers. • Soluble tyrosine protein kinases belong mostly to the src family. The normal src family kinases transmit mitogenic signals from growth factor receptors in focal adhesions. The oncogenically activated forms are overactive and/or insensitive to negative controls. • Soluble serine/theonine protein kinases include Raf-related protein kinases. The oncogenically activated forms are structurally abnormal variants that are constitutively active. • Small G-proteins of the Ras family are mutationally activated in 20 % of all cancers. The oncogenic forms have point mutations which impair the intrinsic GTPase activity: once activated, the Ras protein is locked in the “on” conformation. • Nuclear transcription factors are encoded by many oncogenes, including myc, jun and fos. The myc oncogene (normally activated by mitogens as an early-response gene) is amplified in many tumors.

26.9. Nuclear Proteins in Cancer Abnormal regulation of the G1 checkpoint is common in cancers. Typical defects are: • Overexpression of cyclin D1, the major D-cyclin. Also cyclin A is occasionally overexpressed. • Cdk4, the major catalytic partner of the D-cyclins, is overexpressed in some cancers. • Cdk-inhibitors are inactivated in many cancers, especially INK4a (inhibitor of kinase 4), an inhibitor of Cdk4 that mediates growth-inhibiting stimuli. • The retinoblastoma protein, pRb, is inactivated in many cancers. Also components of the DNA damage response system are abnormal in many cancers: • p53 is mutationally inactivated in at least half of all spontaneous tumors. This is the most common type of abnormality in spontaneous cancers.

492

Inherited Cancer Susceptibility

26.11

• Mdm2 is a protein that inhibits p53 in normal cells. Many of those tumors that have intact p53 overexpress Mdm2. Anti-neoplastic drugs are in trials which disrupt the Mdm2-p53 interaction in cancer cells. Mutational inactivation of the p53-induced Cdk-inhibitor p21 is extremely rare in cancer. Therefore the apoptosis-inducing activity of p53 appears to be more important than its anti-mitogenic effect for tumor suppression. Many oncogenic mutations, for example myc amplifications, lead to apoptosis in cells with an intact p53 system. Cancers lacking p53 or overexpressing Mdm2 have high mutation rates and are sensitive to mutagens, but they are often resistant to anti-tumor drugs because they cannot go into apoptosis.

26.10. Virally-Induced Cancers Virally induced cancer is rare. For specific viral carcinogenesis, the virus has to integrate its DNA into the host cell genome. The integrated viral DNA can stimulate the expression of cellular proto-oncogenes by promoter insertion or enhancer insertion, but some virus have their own oncogenes. Oncogenic retrovirus integrate their own cDNA into the host cell DNA during their normal life cycle. Oncogenic retrovirus have a viral oncogene in addition to the viral gag, pol, and env genes. The oncogene has been hijacked from a host cell during retroviral evolution. Rous sarcoma virus, which causes sarcomas in chickens, contains the src oncogene. It is fully infective, but all other known oncogenic retrovirus are defective and can replicate only if the cell is also infected with another, intact retrovirus. Retroviral cancers are extremely rare in humans. Oncogenic DNA virus integrate their DNA into the host cell genome only by accident. Some DNA virus contain oncogenes that stimulate the proliferation of the host cell during normal infection and can make the cell cancerous if the viral DNA becomes integrated into the host cell DNA. Some strains of human papilloma virus (wart virus) contain oncogenes whose products inactivate p53 and pRb, the products of the major cellular tumor suppressor genes. These viral oncogenes are unrelated to normal cellular genes. Permanently integrated papillomavirus DNA is present in most cervical cancers, many other anogenital cancers and in Kaposi-sarcoma.

26.11. Inherited Cancer Susceptibility A minority of cancers, including 5 % of colon cancers and 5 % of breast cancers, occur in patients with inherited cancer susceptibility (these numbers come from a genetics text book; recent results indicate that at least for breast cancer, the true number is higher). Benign

493

26.11

Biochemistry and Genetics

tumors can also be caused by inherited mutations. Most cancer susceptibility syndromes are caused by an inactivating mutation in a tumor suppressor gene. Susceptible individuals are heterozygous for the gene defect, therefore the cancer susceptibility is inherited as an autosomal dominant trait, normally with reduced penetrance. Somatic cells with the heterozygous mutation grow normally, but a cell that loses the second, intact copy of the gene is likely to become neoplastic. From the cell’s point of view, the mutation that makes it malignant is recessive. Inherited cancer show a number of characteristics that a clinician should keep an eye on: these patients show up with a cancer earlier than the average patient; these patients have more than one focus more often than other patients; there is additional support for suspicion of an inherited cancer if another family member has an otherwise rare cancer. The increased risk of specific types of rare cancers are characteristic for each familial cancer gene, e.g., BRCA1 predisposes for breast and ovarian cancer and more rarely for cancer in colon and prostate, while BRCA2 predisposes for breast cancer, also with some risk in male carriers while ovarian cancer is less common than in BRCA1, and furthermore BRCA2 carriers have a low but increased risk for cancer in prostate, pancreas, throat, esophagus, colon, as well as gall bladder and ducts. Retinoblastoma is a rare tumor of immature retinal cells in infants. In 60 % of the cases, only one tumor is present and there is no family history. In 40 %, multiple tumors are present and/or there is a family history. These patients are born with an inactivating mutation of the retinoblastoma (Rb) gene in all their cells, either as a result of a new mutation (no family history), or inheritance from an affected parent. When a somatic mutation inactivates the second, intact copy of the Rb gene, the cell becomes malignant. This happens in 90 % of those with the inherited mutation (90 % penetrance), and most of the time it happens in more than one cell. Some of the survivors get sarcomas later in life. Homozygous Rb mutations are also seen in sporadic retinoblastoma, and in many bladder cancers, sarcomas, and other malignancies. Patients with Li-Fraumeni syndrome have a high risk of breast cancer, sarcomas, leukemias, brain tumors, and adrenocortical cancer. It is caused by mutations in the p53 gene. Most patients have point mutations affecting the DNA-binding region of the p53 protein. Cells become cancerous when the second copy of the p53 gene is lost and additional oncogenic mutations take place. Breast cancer is caused by inherited cancer susceptibility in 2–5 % of all patients. Inherited mutations in the BRCA1 and BRCA2 tumor suppressor genes cause both breast and ovarian cancer, with a lifetime risk of 80 % (lower in some studies). The tumors of these patients have homozygous inactivation of the tumor suppressor gene. BRCA1 and BRCA2 are also inactivated in a few spontaneous breast cancers but less often than e.g., the APC gene is involved in spontaneous colon cancer. Inherited cancer susceptibility has to be suspected in cases of early-onset breast cancer with a positive family history.

494

Objectives in Brief

26.12

Patients with adenomatous polyposis coli (APC) gradually develop thousands of polyps in the colon mucosa, and one or several of them become malignant sooner or later. The affected gene codes for a membrane protein that may be important for contact inhibition. When the second copy of the gene becomes inactivated, a polyp forms. Additional mutations can create a malignant tumor. Most spontaneous colon cancers also have homozygous APC inactivation, however, few polyps are expected in cases of spontaneous APC. Prophylactic removal of the colon is recommended for established cases of familial APC. Hereditary non-polyposis colorectal carcinoma (HNPCC) is caused by inherited mutations in a gene for post-replication mismatch repair. When the second copy of the gene is inactivated by somatic mutation, the cell develops a mutator phenotype. Somatic mutations accumulate rapidly, until the cell either dies or becomes malignant. Most patients develop colon cancer, but the risk of other cancers is also increased. Several (non-allelic) mismatch repair genes are affected in different patients. Mismatch repair defects are also seen in some spontaneous cancers. Patients with neurofibromatosis have multiple neurofibromas (nerve sheath tumors), cutaneous café-au-lait spots, and iris haematomas (Lisch nodules). The tumors are benign, but some patients develop neurofibrosarcomas. The most common form of the disease (NF-1, von Recklinghausen’s disease) is caused by inactivating mutations in the gene coding for a Ras-GTPase-activating protein, but the pathogenic mechanism is unclear. Homozygous inactivation of NF-1 is seen in malignant tumors of NF-patients, but not necessarily in the benign neurofibromas. Genetic screening for inherited cancer susceptibility is becoming possible, especially for the relatively common breast cancer mutations. A test is already available for a BRCA1 mutation that occurs in 1 % of all Ashkenazi Jews and is responsible for 10 % of breast cancers in this population.

26.12. Objectives in Brief 1. Define the term checkpoint with respect to the cell cycle. Name two cell-cycle checkpoints. 2. Describe the roles of cyclins, retinoblastoma protein (pRb), and mitogens in control of the cell cycle. 3. Give examples of transcription factors, structural nuclear proteins and enzymes whose abundance or phosphorylation state fluctuate during different stages of the cell cycle. 4. Explain the cellular process of apoptosis and its relation to the activity of the p53 protein. Give examples of when apoptosis is likely to occur.

495

26.12

Biochemistry and Genetics

5. List major signal transducing events in the MAP kinase, phospholipase C and PI3K/Akt pathways. 6. Give examples of oncogenes encoding growth factor receptors, GTP-binding proteins, non-receptor tyrosine kinases and transcription factors. 7. Describe the two- hit model for the inactivation of tumor repressor genes, and the mechanism by which the mutations in proto-oncogenes cause excessive cell proliferation. 8. Compare retroviral oncogenes with cellular oncogenes and with the oncogenes of the papillomavirus. Describe how virus can activate cellular proto-oncogenes by promoter insertion and enhancer insertion. 9. Name two molecular lesions that are likely to cause genomic instability and a mutator phenotype in cancer cells. 10. Define the terms hyperplasia, hypoplasia, and metastatic growth. Name two molecular lesions which are likely to cause progression of a cancer cell toward the metastatic state. 11. Describe the multiple-hit theory of carcinogenesis with respect to specific genetic lesions associated with development of cancer of the colon. 12. Name at least three genes which are inactivated in common spontaneous cancers in humans.

496

27. Immunoglobulins and Immunogenetics 27.1. Antibody Structure Immunoglobulins (“antibodies”) account for 20 % of the plasma proteins. Most are γglobulins, but some migrate in the β and α2 fractions. Immunoglobulins are also present in interstitial fluid, exocrine secretions (tears, saliva, bronchi, GI-tract), and on the surface of B-lymphocytes. They are glycoproteins containing 2–12 % carbohydrate. They all can bind specific antigens.

27.1.1. Structure of Immunoglobulin G (IgG) Immunoglobulin G (IgG) consists of 2 light (L) chains (MW 23 000 Da) and 2 heavy (H) chains (MW 50 000 Da). The two H chains are associated with one another in the C-terminal half of the polypeptides. Near their middle they are connected by inter-chain disulfide bonds. Each L chain associates with the amino-terminal half of H chain, both non-covalently and by disulfide bonding. The polypeptides are folded into globular domains, each stabilized by an intra-chain disulfide bond. Each L chain has 2 domains, each H chain 4. The domains at the amino-ends of each chain are variable in different IgG-molecules. The other domains are constant. Each variable domain contains 3 or 4 hypervariable regions. The variable domains are responsible for antigen-binding, the constant domains of the H chains for the “effector functions”, such as stimulation of phagocytosis, complement activation, and placental transfer. Between the first and second constant domains of the H chains there is a small non-globular portion called the hinge region. It is more flexible than the rest of the molecule and accessible to proteases. It contains the disulfide bonds between the H chains. Papain cleaves the H chain in the hinge region, on the amino side of the disulfide bridges, to create two Fab -fragments (ab = antigen-binding) and one Fc fragment (c = crystallizable).

497

27.1.1

Biochemistry and Genetics

"

"

# $$"

!

!

"

% &'!%(

Figure 27.1.: Structure of immunoglobulin G, the most common type of antibody. Figure from [Buxbaum, 2007].

Figure 27.2.: The immunoglobulin fold (PDB-code ifc2) is a sandwich of two anti-parallel β-pleated sheets.

498

Heterogeneity of Immunoglobulins

27.1.3

The Fab fragment binds the antigen, and the Fc fragment is responsible for the effector functions. Immunoglobulin molecules have two antigen binding sites, each formed by the variable domains of one L chain and one H chain. Antigen binding is non-covalent and reversible. The antigen is a foreign molecule of high molecular weight that induces the formation of a matching antibody. The specific portion of the antigen molecule that induces antibody formation and binds the antibody is called the antigenic determinant, or epitope. Antibodies of all classes bind antigens, although their effector functions are different.

27.1.2. Heterogeneity of Immunoglobulins There are 5 classes of immunoglobulins: IgG, IgA, IgM, IgD, and IgE. Each class has its own type of heavy chain: γ-chains for IgG, α for IgA, µ for IgM, δ for IgD and  for IgE. IgG, IgA and IgM have subclasses with slightly different heavy chains: γ1, γ2, γ3, and γ4 for IgG1, IgG2, IgG3, and IgG4, α1 and α2 for IgA1 and IgA2, and µ1 and µ2 for IgM1 and IgM2. There is only one class each for IgD and IgE. There are 2 types of L chains, λ (lambda) and κ (kappa). 2/3 of immunoglobulin molecules in all classes have two κ-chains, 1/3 have two λ-chains. Polymeric forms occur in the IgA and IgM classes: IgA occurs as a monomer (MW 160 kDa) or dimer. Serum IgM is a pentamer (MW 900 kDa). The polymeric forms contain a single J chain (J = joining), which is disulfide-bonded to the H chains. Secreted IgA contains a secretory component, a 70 kDa glycoprotein. IgG 75 % of all immunoglobulins. Placental transfer. Maternal IgG protects the newborn from infections. Weak to moderate ability to fix complement (except IgG4). Has memory: anamnestic response. IgA The major immunoglobulin in external secretions. Secretory IgAis a dimer consisting of two H2 L2 units, a J-chain, and the secretory component. Fights infections on mucosal surfaces, and scavenges antigens before they induce an IgE (allergic) response. IgM Present as a pentamer in plasma. Strong complement binding. IgM levels rise more rapidly than IgG in the course of an infection. Some antigens induce only an IgMresponse but no IgG-response (example: ABO blood group antigens). No anamnestic response. IgD Present in trace amount only. Unknown function. IgE Binds to the surface of mast cells, basophils, and alveolar macrophages. Binding of antigen to cell-bound IgE induces the release of histamine and heparin during allergic responses. IgE is elevated in many allergic patients.

499

27.1.3

Biochemistry and Genetics

Figure 27.3.: Structure of MHC-1 (PDB-code 1qse).

27.1.3. Other Ig Domain Proteins T-Cell Receptors (TCR) T-cell receptors occur in the plasma-membrane of T-cells and are similar to one arm of an antibody. They bind to antigen-loaded MHC-molecules on antigen-presenting cells (all cells for MHC-I and phagocytic cells for MHC-II). Like antibodies, they are composed of two chains. The binding site is at the tip of the molecule, and is formed of by several loops of the protein chains. Other Membrane Immune Receptors: CD4 and CD8 membrane-bound receptors also contain repeats of the immunoglobulin fold. These receptors act as co-receptors and stabilize the interaction between TCR and antigen-loaded MHC. They are unique markers for T-cells (CD4+ in Th - and CD8+ in Tk -cells). Classes of cellular adhesion molecules (CAMs) also contain multiple repeats of the immunoglobulin fold which function to form cellular contacts by dimerization. Cadherins

500

The Major Histocompatibility Locus (MHC)

27.2.1

D. Clinical Methods Figure 27.4.: How to perform an ELISA. The test can be used to detect either antigens or antibodies. 1. Enzyme-linked Immunosorbent Assay

a. Indirect ELISA measure the amount of specific antibody b. Sandwich ELISA measures the amount of antigen in a sample also contain immunoglobulin-like repeats. Fibronectin, an extracellular matrix protein, also contains repeats of the immunoglobulin II. IMMUNOGENETICS fold.

A. The Major Histocompatibility Locus (MHC) Clinical Methods The human major histocompatibility complex (MHC) or human leukocyte antigen (HLA) region is of greater than 4 Mb of DNA (~0.1 % of the genome). Location: Enzyme-linked . Short arm ofImmunosorbent chromosome 6 Assay at 6p21.3

There are greater than 200 genes located in the MHC and ~ 40 % of these genes have immune related functions. Traditionally, these are discussed as class I, class Immunogenetics II and class III genes with the sequence centromere- II - III - I - telomere. 27.2.

27.2.1. The Major Histocompatibility Locus (MHC) The human major histocompatibility complex (MHC) region is of greater than 4 Mb of DNA (≈ 0.1 % of the genome). Location: Short arm of chromosome 6 at 6p21.3

260

501

27.2.1

Biochemistry and Genetics

Figure 27.5.: Principle of immuno-assays. In an indirect test, labeled antigen competes with sample antigens for a limited amount of antibodies. The more (unlabeled) sample antigen is present in the sample, the less signal is measured. A sandwich assay uses two different antibodies, the capture antibody is immobilized on the substrate and binds the antigen. The amount of antigen is then measured using a labeled detection antibody, that binds to a different epitope on the antigen. In a direct test, labeled antibody is used to detect immobilized antigen. In an indirect test, the detection antibody itself is not labeled, but the amount of bound antigen is measured by labeled secondary reagent (antibody against the Fc -part of Ig, protein A, protein G or similar). Figure from [Buxbaum, 2007].

FRPSHWLWLYH

GLUHNW

502

VDQGZLFK

LQGLUHNW

The Major Histocompatibility Locus (MHC)

27.2.1

Figure 27.6.: Organization of genes in the MHC-region of chromosome 6.

There are greater than 200 genes located in the MHC and ≈ 40 % of these genes have immune related functions. Traditionally, these are discussed as class I, class II and class III genes with the sequence centromere- II - III - I - telomere.

Class I region Highly polymorphic functional classes : HLA-A, HLA-B, HLA-C. Class I molecules are expressed at the cell surface of most tissues. Functions: • antigen presentation to cytotoxic T lymphocytes (CTLs) • signal of “electrically silent neurons” to CTL surveillance system • No expression at materno-fetal interface facilitates survival of fetal tissues • HLA-C involved in target recognition by natural killer cells • HLA-E, -F, -G have more selective tissue distributions • HLA-G is expressed on the maternal trophoblasts effecting fetal tolerance • HFE gene implicated in hereditary hemochromatosis

503

27.2.1

Biochemistry and Genetics

Figure 27.7.: Structure of MHC-1.

• Other non-immune genes include those of zinc-finger proteins, ubiquitin-like proteins, olfactory receptor family, and butyrophilin family genes Class II region Class II molecules are encoded in a region of about 800 kb of DNA with approximately 20 genes. [Structure below has membrane segment removed.] • HLA-DP, HLA-DQ, and HLA-DR are expressed on antigen presenting cells. They present peptides to T helper cells. • Many pseudo-genes of paired HLA-DP and HLA-DQ are also present • HLA-DM proteins participate in transfer of peptides onto class II molecules within intracellular vesicles. • Some ABC transporter superfamily genes are present: These genes encode proteins which transport a variety of substances across membranes: oligopeptides, proteins and ions. TAP1 and TAP2 are genes for translocases found in the ER membrane. TAP function is required for Class I antigen presentation, suggesting intracellular linking of their functions. • LMP2 and LMP7 genes encode proteins of the proteosome complex.

504

The Major Histocompatibility Locus (MHC)

27.2.1

Figure 27.8.: Structure of MHC-II.

• LMP and TAP genes, as well as some class I molecules are induced by gamma interferon (IFNγ), an immune-stimulator. Class III region 1.1 Mb segment of DNA between class I and class II regions contains approximately 70 genes. One internal DNA segment contains a duplicated gene cluster with complex and overlapping gene arrangements. Many class III genes encode immune-related proteins: • PBX2 is a homeodomain protein which participates in hematopoiesis • RAGE is ’receptor for advanced glycation end-products of proteins’. This protein mediates monocyte migration and activation in response to advanced glycation endproducts. Binding of AGE’s to RAGE in the endothelium induces NF-κB and increased vascular permeability. RAGE could be a link in diabetes to vascular complications because of increased AGE formation in diabetics. • G15 (hLPAATα) encodes an enzyme associated with prostaglandin metabolism, and important inflammatory mediator. Prostaglandins play important roles in production of pain and fever, the regulation of blood pressure, induction of blood clotting, control of several reproductive functions and regulation of the sleep-wake cycle. • Complement components C2 and C4 participate in the classical complement pathway, stimulating yeast, bacterial and viral recognition with mannan-binding proteins. The C4 gene loci are highly polymorphic. Partial deficiency of C4 is associated with increased susceptibility to systemic lupus erythematosus (SLE), scleroderma and primary biliary cirrhosis.

505

inflammatory and immunomodulatory activities as well as being involved in tumor cachexia.

27.2.2

B. Immunoglobulin Gene Structure Biochemistry and Genetics Exons of immunoglobulin genes are arranged in a manner that makes possible the generation of many unique immunoglobulin genes, and transcript splicing patterns. Shown below is a diagram with the unique segments arranged Figure 27.9. in tandem copies.

Structure of the IGH Ig heavy chain gene at 14q32 V region (900 kb)

DJC region (350 kb)

Telomere

~100 VH segments

Centromere

26 DH seg’s

11 CH seg’s

9 JH seg’s Antigen binding diversity is encoded here. Selection of unique VH segments occurs in B cell maturation.

Antibody Class (IgM, IgD etc.) is encoded here. Unique CH segments encode class switching gene segments. 263

• The G9 gene encodes a sialidase important for lysosomal function. Hyposialydation of cell surface proteins on T cells appears to be required for normal T-cell function, and higher sialydation may result in some types of autoimmune disease. • Heat shock proteins Hsp70-1 and -2 genes encode proteins induced by high temperature. • The TNF ligand superfamily of cytokines are also expressed: TNF, LTα and LTβ. TNF is produced by a variety of cells and exhibits numerous inflammatory and immunomodulatory activities as well as being involved in tumor cachexia.

27.2.2. Immunoglobulin Gene Structure Exons of immunoglobulin genes are arranged in a manner that makes possible the generation of many unique immunoglobulin genes, and transcript splicing patterns. Shown below is a diagram with the unique segments arranged in tandem copies.

506

Immunoglobulin Gene Structure

27.3.1

Figure 27.10.: Somatic recombination leads to a large number of different antibodies.

Generation of Antibody Diversity Biosynthesis of Immunoglobulins Immunoglobulins are secreted by plasma cells which are the descendants of B-lymphocytes (B-cells). 15–30 % of lymphocytes are B-cells. Tlymphocytes do not produce antibodies but have T-cell receptors on their surface instead, with idiotypes similar to immunoglobulins. Therefore both B-cells and T-cells can recognize antigens. B-cells do not secrete immunoglobulins, but they possess surface immunoglobulins in their plasma membrane: Initially only IgM, later IgD, then IgG, IgA or IgE. These successively expressed antibodies have the same variable domains, but the constant domains of the Hchain are different. This process is called class switching. Important: each B-cell makes an antibody of only one antigen-binding specificity. We have millions of different antibodies because we have millions of B-cells, each making its own antibody. The differentiation of the B-cell into a plasma cell requires the binding of an antigen to the surface immunoglobulin and stimulation from helper T-cells. Only those B-cells that encounter their antigen become plasma cells. This is called clonal selection.

507

27.3.2

Biochemistry and Genetics

27.3. MHC (HLA) and Clinical Risk of Disease 27.3.1. MHC Polymorphisms and Disease Risk The genes of the MHC are highly polymorphic possibly due to selective pressure of regional pathogens. More than 200 allelic variants are known for some highly polymorphic genes, and some of these alleles are associated with the incidence of disease.

Diabetes Mellitus Europeans with Type I have a higher frequency of (96 vs 45 % expected) of the DR3 or DR4 alleles present at the Class II DRB1 locus on 6p21.3 (in one copy) and a higher frequency had both alleles (38 % vs. 3 % expected) Also the loci DQA1 and DQB1 may be predisposing/protecting. The mechanism for predisposition and protection are not understood.

Rheumatoid arthritis (RA) High risk is associated with specific alleles within the DR4 locus: Two alleles, DRB1∗0401 (previously called Dw4) and DRB1∗0404 (previously called Dw14), primarily account for the DR4 association with disease in Caucasians. These alleles all have in common the “shared epitope”, suggesting a specific protein sequence is associated with susceptibility to RA.

Ankylosing spondylitis (AS) Thirty percent of the susceptibility to ankylosing spondylitis appears to be encoded by HLA-B27 genes

Common variable immunodeficiency (CVID) and IgA deficiency (IgAD) susceptibility and resistance are most closely associated with the genes within the HLA DR-DQ regions.

27.3.2. Familial Immune Disorders Immunoglobulin subclass deficiencies Most of these are benign, due to overlapping functions within immunoglobulin subclasses. Immunoglobulin A deficiency (IgAD) is the most common, with an incidence 1 in 500. IgAD is multifactorial with HLA associated risk. Chronic variable immunodeficiency (CVID) affects approximately 1 in 10 000 to 100 000 individuals. The patients display a marked reduction in serum levels of both IgG and IgA. In half of the patients, IgM is also reduced. Patients are at much greater risk for recurrent bacterial infections.

508

Objectives in Brief

27.4

Bruton agammaglobulinemia X-linked inheritance. Incidence is 1/100 000. Characterized by failure to produce mature B lymphocyte cells and associated with a failure of Ig heavy chain rearrangement. The defect in this disorder resides in Bruton tyrosine kinase (BTK, also known as BPK or ATK), a key regulator in B-cell development Decreased IgM, IgG and IgA in serum. Less than 1 % of B cell number. DiGeorge Syndrome (Congenital Thymic Dysplasia) Most cases result from a deletion of chromosome 22q11.2. The spectrum of immune dysfunction is wide. Defects are due to developmental abnormalities of the thymus, cardiovascular and endocrine organs. Adenosine Deaminase Deficiency Autosomal recessive form of severe combined immunodeficiency disease (SCID-ADA). Toxic metabolites of adenosine accumulate with the immune cells. These metabolites inhibit normal lymphocyte proliferation. Patients have reduced function of both B and T lymphocytes, non-functioning circulating immature T lymphocytes, and hypogammaglobulinemia. Plasma Cell Dyscrasias These are neoplastic diseases which are also called paraproteinemias or monoclonal gammopathies. Patients show a sharp peak in the γ-globulin region (sometimes β or α2) on serum electrophoresis. This peak (“paraprotein”) is a homogeneous immunoglobulin, produced by a single plasma cell clone. The remainder of the γ-globulin fraction is depressed. Types: Multiple myeloma: Malignant proliferation of a plasma cell clone in the bone marrow. With anemia, bone lesions, bone pain, pathological fractures, recurrent infections. The paraprotein is most commonly IgG (50 %) or IgA (25 %). Some patients overproduce only L-chains which are excreted in the urine: Bence-Jones protein. Waldenstroms macroglobulinemia: Overproduction of IgM, malignant, with blood hyperviscosity. Benign monoclonal gammopathy Present in 5 % of persons above age 50 and 8 % of those over 70. May occasionally progress to multiple myeloma.

27.4. Objectives in Brief 1. Describe the general structure of immunoglobulins (Igs) and state the nature and consequences of antigen-antibody binding. 2. Name the most important functions of the five major classes of Igs. 3. Describe the Major Histocompatibility Complex (MHC).

509

27.4

Biochemistry and Genetics

4. Explain the importance of the MHC in human disease risk. 5. Describe Immunoglobulin gene structure. 6. Explain the terms class switching and clonal selection in relation to IgG gene structure. 7. Discuss the mechanism of cellular generation of antibody diversity. 8. Name the principal types of monoclonal gammopathy, and define the term “BenceJones protein”.

510

28. Inherited diseases of metabolism 28.1. Introduction 28.1.1. Significance of inherited diseases of metabolism inherited disease of metabolism (IDoM) are individually rare, with 1 case per several thousand to several hundred thousand births. However, since there are 350 known diseases of this type, together they contribute significantly to infant morbidity and mortality. About 1 in 2500 births has some form of inherited metabolic disease, and you are not unlikely to encounter some cases during your career. The point is, however, that you do not know yet which ones it will be. IDoM usually are inherited in an autosomal recessive fashion. This means that they may be enriched in inbred populations (Mennonites in Pennsylvania, Ashkenazi Jews) and that founder-effects may be observed (e.g. tyrosinemia in Quebec). Exceptions from the autosomal recessive inheritance (X-linked, autosomal dominant or dominant negative) will be noted. Many important diseases (diabetes, auto-immunity) have a genetic component, but also require environmental factors to become manifest. Clinically IDoM are very variable, onset of symptoms may be within 24 h after birth, or it may occur in mature people. Symptoms may develop suddenly, or slowly and insidiously. Even patients with the same mutation, for example from the same family, may show very different clinical pictures. Some IDoMs may be phenocopies of other diseases, that is they present clinically like some other (non-inherited) disease. The first such pair described in the literature were urea cycle defects, that may present like Reye-syndrome. As a consequence, IDoM tend to be under-diagnosed.

28.1.2. Mechanism of IDoM Inherited diseases of metabolism usually occur because of a defect in the gene for an enzyme. This may result in: inappropriate transcription to few/many copies of mRNA

511

28.1.2

Biochemistry and Genetics

splicing defect incorrect mRNA produced (e.g. spinal muscle atrophy). About 15 % of single nucleotide mutations interfere with splicing. translation e.g. nonsense-mutations: inappropriate Stop-signal. May be the result of a splicing defect, if an intron is maintained that contains a stop-signal. folding protein folds to slowly and is destroyed by ERAD/proteasome (e.g. Cystic fibrosis). Those enzyme molecules that manage to fold may function perfectly fine. amyloid formation improperly folded protein accumulates and interferes with cellular function (e.g. α1 -antitrypsin in liver). defective protein sequence or structure does not allow function. In oligomers may lead to dominant negative inheritance (e.g. aquaporin in diabetes insipidus). regulation of activity site for post-translational modification destroyed or protein becomes independent of modification (e.g. oncogenes). regulation of survival time too low/high concentration of enzyme. When an enzyme is defective, the following problems may occur: • lack of product • accumulation of substrate • conversion of substrate to toxic substances by other enzymes • gain of function – protein does something it shouldn’t (→ autosomal dominant!) Sex-linked diseases X-linked inheritance The X-chromosome is unusual, in that ~ have two, | only one copy. To compensate for the increased gene dose, ~ inactivate one of the two X-chromosomes as Barr-body (Lyons-hypothesis. Inactivation occurs early in embryonic development in such a way, that some of the cells inactivate the X-chromosome from the father, others that from the mother. This pattern is than fixed during development, so that ~ are a somatic mosaic. Most heterozygous ~ with a recessive mutation on the X-chromosome have the correct copy active in about 50 % of their cells, and appear normal, although they are carriers (can transmit the defect gene to their offspring). Some however are mildly affected (manifestation), as too few cells in their body make the correct gene product (variable penetrance). | carrying such a mutation are hemizygote and fully affected, as they have no correct copy to compensate. The most well known example for this mode of inheritance is hemophilia A. If 1 : q is the frequency of the defect gene in the population, then 1 : q |will show the disease, but only 1 : q 2 of ~ with two defect copies will be affected.

512

Newborn screening

28.1.3

The number of Barr-bodies in a cell is the number of X-chromosomes minus one. Xinactivation is incomplete, some genes are expressed from all copies of the X-chromosome. Hence patients with extra X-chromosomes (47,XXX or 47,XXY) show defects. A few X-linked diseases are dominant, e.g., Rett-syndrome (OMIM #312750), the second most common cause of inherited mental retardation in ~ after trisomy 21. In those cases affected | die in utero (unless 47,XXY or the like), only ~ with one defect copy express the disease. As affected ~ hardly ever reproduce, most cases are the result of new mutations (usually in the paternal germline). Sex-limited diseases can only occur in one sex (e.g. testicular or ovarian defects). Sex influenced diseases are more common and/or more sever in one sex than the other. For example, male-pattern baldness results from a hypersensitivity of hair follicles to the androgen dihydrotestosterone. The disease is dominant in |, recessive in ~ due to different hormone levels.

28.1.3. Newborn screening In many cases, damage by IDoM can be limited by an appropriate diet, which supplements compounds that the patient can not synthesize, or limits nutrients that they can not metabolize. Such dietary management in many cases may however only prevent the worsening of damage; such damage that has already occurred, especially to the nervous system, can not be cured. Given the suffering involved for patients and their families, and the high costs incurred by society, screening of newborns for certain IDoM is required by law in many developed countries. The catalogue of diseases, for which you are obliged to screen, varies between jurisdictions, but most commonly includes the following: • phenylketonuria • galactosemia • hypothyroidism • mucopolysaccharidoses There are some screening tests, that are inexpensive and so simple to perform that they may also be done by community nurses or midwifes, it involves nothing more than pouring a few drops of reagent solution onto a used diaper and observing for color changes. Their lack of specificity is an advantage in this case, as they react to any of several severe conditions: FeCl3 phenylketonuria, tyrosinemia, maple syrup urine disease, alcaptonuria and ketonuria. Interference by salicylates and phenothiazines.

513

28.1.3

Biochemistry and Genetics

Reducing substances glucose, galactose, fructose, lactose, sialic acid. Interference by cephalothin and ampicillin Nitroprusside sodium ketones, cystine, homocysteine Azure A mucopolysaccharides Any positive result in these tests needs to be followed up with more specific investigations, which however require a specialized laboratory. Blood samples from a heel-prick may be used for screening, either fresh or dried on absorbent paper. Common tests include: • PKU, MSUD, homocystinuria, tyrosinemia • MCAD! deficiency, biotinase deficiency, galactosemia • adrenal hyperplasia, hypothyroidism • sickle cell disease The most modern technology for screening uses liquid chromatography coupled to multidimensional mass spectrometry (LC/MS) to determine the identity and concentration of virtually any compound in a sample. Any unexplained deviations from normal need followup. Although the initial instrument costs are very high, this method allows high-throughput screening of samples with minimal staff involvement. Given all the fancy technology available to physicians nowadays one should not forget that careful observation and a thorough physical exam of a patient can alert an astute physician to the possibility of an IDoM. The following signs should raise suspicion: • lethargy, convulsions, hypotonia • hepatomegaly, also kardio- or splenomegaly • acidemia, increased anion gap • hyperammonemia • hypoglycemia • unexplained vomiting • elevated liver enzymes • unusual color, smell or structure of hair, eyes, skin, stool or urine

514

28.1.3

Newborn screening

Figure 28.1.: Liquid chromatography with multi-dimensional mass spectrometry is a modern technology used to determine metabolites in urine or deproteinized serum. Molecules in the sample are separated from each other by liquid chromatography, the effluent from the column is injected into an ionizer, which turns the dissolved sample molecules into single ions in vacuum. Following a high electric potential the molecules fly through the first analyzer, which determines their molecular mass. Ions of a given mass then enter a collision cell, where they bump into He atoms. The force of the collisions makes the ions break into fragments. Different bonds have different stabilities, hence the breaking points are specific for each compound. The molecular weights of the fragments are then determined in a second analyzer, giving a “fingerprint” spectrum. Thus from LC/MS/MS you get for each compound in the sample the chromatographic position and concentration, molecular weight and fragment spectrum. This is sufficient to reliably identify and quantify (almost) all compounds in a sample. Figure from [Buxbaum, in press].

Chromatographic column (RPC, IEC)

Sample injector

Mobile phases

Chromatographic Peak identification quantitation

sample identification

He gas (low pressure)

Photometer

Ionizer dissolved sample -> ion stream in vacuum

Data base

Internet

Analyzer determines molecular mass of sample ions

Collision cell fragments sample ions of given molecular mass

2nd Analyser determines molecular mass of fragments (fingerprint)

515

28.2.1

Biochemistry and Genetics

Signs may worsen in stress (infection, exertion). Some IDoM are associated with characteristic smells of urine, sweat or breath: Disease

Pathway

Description

diabetic ketoacidosis hawkinsinuria isovaleric acidemia maple syrup urine disease oast house syndrome methylmalonic aciduria phenylketonuria propionic acidemia trimethylaminuria tyrosinemia I urea cycle defect

Glc Tyr Leu Leu, Ile, Val Met odd-chain fa, aa Phe Ile, Val, Thr, Met choline, carnitine Tyr amino acids

acetone, fruit chlorine-like sweaty feet caramel drying hops ammonia mouse urine ammonia fish cabbage ammonia

28.2. Cytosolic enzymes Carbohydrate, amino acid and nucleotide metabolism occur mostly in the cytosol. Most of the following diseases you will have encountered already in their respective pathways.

28.2.1. G6PDH deficiency – Favism G6PDH is the first committed step in pentose phosphate pathway, which supplies ribose (for nucleic acid synthesis) and NADH + H+ . The later protects the cell against oxidative damage, it is required for the regeneration of oxidized glutathione. Especially in erythrocytes, which are exposed to a particularly high oxidative stress, the pentose phosphate pathway is the only major source of NADH + H+ (only anaerobic glycolysis!). Reduction of G6PDH activity in heterozygotes leads to an advantage in malaria-infested regions (Mediterranean, Africa, Asia). The higher redox-potential and shorter life time of the erythrocyte limits growth of the blood-stage of the malaria parasite. Favism is inherited in an X-linked recessive manner, and therefore protects mainly ~. Affected |(about 100 Mio worldwide) appear usually normal, but will suffer an acute hemolytic crisis after ingestion of substances that lead to the formation of reactive oxygen species. Classically, this includes broad (fava) beans (Vicia faba L.), which contain divicine as offending ingredient. Several classes of pharmaceuticals also have members that can precipitate such a crisis, which you may remember by the acronym A4 : analgetics, antipyretics, antimalarials and antibiotics.

516

28.2.3

Galactosemia

Figure 28.2.: Galactosemia is caused by the inability to convert ingested galactose into glucose. The excess galactose is then converted to galactitol and galactonate instead. O

O

O

HO OH

OH HO OH HO

GalDH (via lactone)

aldolase

OH

reductase

HO

HO OH HO

OH

OH Galactonate

OH

HO

OH

Galactose

Galctitol

Gal-1-P uridyl transferase (type I, 1 : 50,000) Gal-kinase

(type II, 1 :100,000)

UDP-Gal-4 epimerase

(type III, rare except Japan)

O OH HO OH HO OH Glucose

During a crisis, damaged hemoglobin will precipitate forming Heinz-bodies, which are visible in a stained blood smear. That results in membrane damage, damaged erythrocytes are then removed by the spleen. Heinz-body anemia also occurs in other diseases with hemoglobin damage (α-thalassemia, poisoning...).

28.2.2. Galactosemia If ingested galactose can not be converted to glucose (due to failure of any one of the three enzymes involved), it will be converted to galactitol and galactonate instead by other enzymes. Those increase the osmotic pressure in the eye lens, leading to swelling, which in turn interferes with membrane integrity. The influx of Na+ from the interstitial fluid leads to further swelling, and finally apoptosis. The result is cataract formation. Other cell types are also affected, leading to difficulty feeding, diarrhea, lethargy, hypotonia, jaundice, hepatomegaly, mental retardation, verbal dyspraxia (difficulty), motor abnormalities and ovarian failure in ~. Management is mainly by Gal-free diet, which can reverse early cataracts, but not the other problems. In addition, disease progression is only slowed (albeit significantly), since our body produces Gal on its own.

517

28.2.5

Biochemistry and Genetics

28.2.3. Errors of fructose metabolism There are three IDoM in fructose metabolism, of which hereditary fructose intolerance is the most dangerous: hereditary fructose intolerance (HFI) is caused by fructoaldolase (aldolase B) deficiency in liver, kidney, and small intestine. It is asymptomatic until Fru, saccharose or sorbitol is ingested, usually after weaning. The disease results in poor growth, liver and kidney damage and, potentially, sudden death. In most cases the disease is self-limiting, because affected kids learn to avoid foods that make them feel sick. Treatment: Fru, Saccharose and sorbitol-free diet, then normal development. Fructosuria is caused by hepatic fructokinase deficiency. Since the fructose is not taken up efficiently into hepatocytes, it is excreted in urine. No pathology, hence no treatment is required. Cave: since Fru is a reducing sugar (spontaneous conversion to Glc, especially under alkaline conditions) fructosuria may be mistaken for diabetes mellitus in urinalysis. Fru-1,6-bisphosphatase deficiency prevents gluconeogenesis and results in exertional or fasting hypoglycemia + acidemia (lactate, pyruvate, ketone bodies). Management involves a Fru-free diet and frequent meals. Under those conditions, development is normal.

28.2.4. Lactase persistence/restriction Lactose in mammal sucklings is split to Gal + Glc by lactase on the brush-border membrane of the small intestine. Expression of lactase is reduced after weaning to 5–10 % of suckling levels, in humans that usually happens at age 3–5 a. In certain populations however, which have a tradition of shepherding, mutations in the cis-acting (=DNA) elements that regulate lactase mRNA transcription result in expression of this enzyme throughout life. The ability to utilize milk from animals as additional food has arisen several times independently during human history (convergent evolution). Although lactose restriction is normal (and indeed observed in ≈ 90 % of humans), rather than a disease, consumption of milk or milk products by such individuals has drastic and unpleasant consequences: The undigested lactose is fermented by colonic bacteria, resulting in flatulence, abdominal pain and diarrhea. Treatment in such cases is supportive, symptoms vanish on their own after a couple of hours. Other mammals too lose their ability to digest lactose after weaning, therefore please do not feed milk to adult cats!

518

Glycogen storage diseases

28.3

28.2.5. Amino acids Errors of amino acid metabolism have been discussed in chapter 22 and will not be repeated here.

28.3. Glycogen storage diseases All glycogen storage diseases are autosomal recessive, except VIII, which is X-linked. They affect one of several isoforms of enzymes and are therefore tissue specific. Mostly affected are liver (hepatomegaly, fasting hypoglycemia) and muscle (hypotonia). Numerical nomenclature of glycogenoses in the literature is confused, we follow OMIM. Most, but not all, glycogenoses affect cytosolic enzymes. 0a defect in liver glycogen synthase GYS1. Hypoglycemia, high blood ketones, increased free fatty acids and low levels of alanine and lactate in fasting, hyperglycemia after meals. 0b defect in muscle glycogen synthase GYS2. Cardiomyopathy and exercise intolerance with absence of muscle glycogen. Ia (von Gierke) Glc-6-Pase defect in the lumen of the ER. Severe hypoglycemia after 2– 4h of fasting since no blood Glc from liver glycogen. Hyperlipidemia and xanthoma, liver + kidney damage, gout, liver adenomas → carcinomas. Treatment: uncooked corn-starch. Ib defect Glc-6-P transport from cytosol into ER. Slow growth, small, hyperlipidemia and xanthomas, hepatomegaly, liver adenoma, neutropenia. Treatment: GM-CSF, uncooked corn-starch II (Pompe) acid maltase (α-1,4-glucosidase) in lysosomes of all organs. Infantile form severe cardiomegaly and death by 3 a from cardiomegalia glycogenica by inactive enzyme. Adult form by reduced enzyme conc: slowly progressive muscle hypotonia (wheel chair, respiratory support, sphincter), vascular damage. Treat with high protein / low carb diet, infusion of recombinant glucosidase precursor. IIIa (Cori-Forbe) Glycogen debranching enzyme defect in muscle + liver. Hepatomegaly, hypoglycemia, growth retardation, progressive skeletal myopathy, cardiomyopathy, fasting hypoglycemia. IIIb like IIIa, but liver only. IV (Anderson) defect in branching enzyme leads to long, unbranched, insoluble glycogen that precipitates in liver. Hepatomegaly and liver cirrhosis, death by age 5 a.

519

28.4

Biochemistry and Genetics

V (McArdle) defect in muscle phosphorylase. Exercise intolerance and muscle cramps, no Cori-cycle, rhabdomyolysis may lead to renal failure. Often appears only in early adulthood. Treatment: Glc before exercise, or avoid strenuous exercise. VI (Hers) defect in liver phosphorylase. Mild to moderate hypoglycemia, mild ketosis, growth retardation, and prominent hepatomegaly. Good prognosis. VII (Tarui) defect in muscle (M) phosphofructokinase. Exercise intolerance with cramps and possibly rhabdomyolysis and myoglobinuria, hemolysis as the same isoform is also expressed in erythrocytes. Gout as Fru-6-P goes through hexose monophosphate pathway: PRPP ↑, AMP is also increased. Compensatory increase of 2,3-DPG: O2 affinity ↑. Glucose intolerance. Prognosis depends on remaining enzyme activity (or replacement by L-form in erys). VIII defect hepatic phosphorylase kinase (X-linked). Hepatomegaly, growth retardation, elevated liver enzymes, hypercholesterolemia, hypertriglyceridemia, and fasting hyperketosis. Symptoms resolve in adolescence. Treatment with dextrothyroxine (D-T4) if required. XI (Fanconi-Bickel) defect glucose transporter GLUT2. Decreased Glc (+Gal) uptake/release by liver, Glc sensing in pancreas, re-uptake in renal tubules. Growth failure, rickets, osteoporosis, dwarfism, hepatomegaly, moon-shaped face, and fat deposits on shoulders and abdomen, fractures and pancreatitis. Glucose, aa, phosphate, protein and uric acid in urine increased, metabolic acidosis. Treatment: antiketogenic diet; water, electrolyte and vitamin D supplementation.

28.4. Lysosomes Lysosomes are required for the breakdown of molecules (intracellular and extracellular) that are no longer required. Failure of any lysosomal enzyme leads to accumulation of its substrate inside the lysosome, this interferes with cellular function. 50 such lysosomal storage diseases have been described. The main problems occur with breakdown of mucopolysaccharides (from the extracellular matrix) and sphingolipids (from the cell membrane). I-cell disease affects all lysosomal enzymes. Several attempts of treatment of lysosomal storage diseases have been published: Bone marrow replacement can slow the progression of the diseases, since the amount of circulating substrates is reduced. This limits uptake into the defective own lysosomes. However, there is significant morbidity and mortality associated with this expensive procedure.

520

28.4.1

I-cell disease

Figure 28.3.: I-cell disease is caused by the inability to add a phosphate group to a mannose group in the sugar-tree of lysosomal proteins. The Man-6-P acts as a signal for transport from the trans-Golgi to the lysosome. Asn

NH

NH

NH

GlcNAc

GlcNAc transport

GlcNAc Man Man

Asn

Asn

Man

Man

GlcNAc transport

GlcNAc Man

Man

Man Man

Man Man

Man GlcNAc Gal

Glycoprotein (core oligosaccharide) cis-Golgi

Glycoprotein (high-Mannose type) ER

GlcNAc-Phosphotransferase

UDP

I-cell disease

UMP Asn

GlcNAc

Gal

Gal

Glycoprotein (complex type) trans-Golgi GlcNAc

Asn

GlcNAc

NH

GlcNAc

GlcNAc

GlcNAc + transport to trans-Golgi

GlcNAc

Man P

Man

GlcNAc

NH

GlcNAc

Man Man

plasma membrane or exocytosis

Man Man

Man

export

Man

+ addition of sugars

Man

+ trimming Man

Fuc

GlcNAc

binding to Man-6-P receptor enrichment in clathrin coated vesicles transport to lysosome

Man

Man Man

P

Man Man

Man Man

Enzyme replacement therapy recombinant enzyme is infused into the patient, binds to cellular Man-6-P receptors and undergoes endocytosis. That way, the enzyme ends up in the lysosome, where it can do its work. Problem: Blood/brain barrier limits effect against CNS damage (may be overcome by high dose: current phase I/II trial). Somatic gene therapy The defective gene is replaced by a working copy in the affected cells. Because of fatalities encountered, all human experiments on gene therapy are currently on halt.

28.4.1. I-cell disease I-cell disease is caused by the failure to phosphorylate Man in the sugar tree of lysosomal enzymes in the cis-Golgi. The Man-6-P group normally acts as a sorting signal for transport from the trans-Golgi to the lysosome. Thus all lysosomal enzymes end up in the default pathway (export out of the cell), rather than in the lysosome, which is rendered

521

28.4.2

Biochemistry and Genetics

non-functional. This results in the formation of inclusion bodies (Name!). Exported lysosomal enzymes do not cause damage to the body, since the pH in the interstitium (7.4) is far away from that in the lysosome (5), so they are inactive. I-cell disease is characterized by abnormal bone + joint development → dwarfism, hepatosplenomegaly, heart valve defects, failure to grow and develop motor skills, corneal clouding and death by age 7 a (congestive heart failure or respiratory tract infection).

28.4.2. Mucopolysaccharidoses and sphingolipidoses

522

28.4.2 Mucopolysaccharidoses and sphingolipidoses

Figure 28.4.: Breakdown of dermatan sulphate in lysosomes GalNAc

GlcUA

S GalNAc

IduA-S

HO HO

O O H

C H2 H

H

H

(X-linked)

H2O SO4

2-

O O H

C H 2

H

HO O S

HO

H

H

O O H

C H2 H

H

H

H

NAc

O

H2O

SO4

2-

HO

HO

MPS IV: Maroteaux

H OH H OH

O O H

H

C H2

H

H

H

H

OH

OH

OH

H2O

HO

HO

GalNAc

β-hexosamidase

H

NAc

H

H

H

NAc

O O H

O

O H

H

C H2

H

HOOC

HO HO

GalNac-4-sulphatase

H

NAc

H

H

H

OH

H

Breakdown of dermatan sulphate

HO

HO

O H

O

O H

H

C H2

H

HOOC IduA

HO O S

α-L-iduronidase

H 2O

MPS I: Hurler (complete) Scheie (partial)

H

H

OH

H

NAc

O O H

OH

H

NAc

H

H

H O OH

OH

H COOH

H

O

O H

H

H

O H

H

C H2

H

HOOC

HO

HO

MPS II: Hunter

OH

H

β1-3

H

NAc

O O H H OH

S

α1-3

H

NAc

H

H

H O O

Iduronat sulphatase

OH

H COOH

H

O

HOOC H

C H

H

O H

H

2

O H

β1-4

HO O S

HO H

O O H

C H2 H

H

H

H

H

H2O

MPS VII: Sly

OH

H

OH

H

NAc

O O H

OH

HOOC H

GlcUA

β-Glucuronidase

HO

O O H

C H2 HO H

H

OH

NAc

H

H

523

28.4.2

Biochemistry and Genetics

Figure 28.5.: Breakdown of heparan in lysosomes S GlcNS

Heparan

GlcUA

-

COO O OH

H2C OH O OH O

OH NH

O

GlcUA

H2C O S O OH

COO O OH

NH

α1-4

GlcNAc

-

O

O

H2C O S O OH O NH

α1-4

C O

OH

β1-4

CH3

C O

OH

β1-4

S

S

GlcNAc

CH3

S

Heparan sulphamidase

H2O

Sanfilippo A SO4

2-

-

COO O OH

H2C OH O OH O

OH

O

COO O OH

O NH

O

H2C O S O OH O NH C O

OH

CH3

C O

OH

NH2

-

H2C O S O OH

CH3 Acetyl-CoA

N-acetyl transferase Sanfilippo C

CoA -

H2C OH O OH O

OH

COO O OH

O

COO O O

O

H2C O S O OH O

OH

NH

NH

C O

OH

CH3

C O

OH

NH

-

H2C O S O OH

CH3

C O CH3

N-acetylglucosamidase

H2O

Sanfilippo B GlcNAc

-

HO

COO O OH

O

-

H2C O S O OH

COO O OH

O NH

O

H2C O S O OH O NH C O

OH

CH3

C O

OH

CH3

β-glucuronidase

H2O

Sly (MPS VII) GlcUA

HO

O NH C O CH3

524

-

H2C O S O OH

COO O OH OH

O

H2O

H2C O S O OH

SO4

2-

O NH C O CH3

HO

-

H2C OH O OH O NH

N-acetylglucosamine-6sulphatase

C O

Sanfilippo D

CH3

COO O OH OH

O

H2C O S O OH O NH C O CH3

28.4.2

Mucopolysaccharidoses and sphingolipidoses

Figure 28.6.: Breakdown of chondroitin- and keratane sulphate in lysosomes Proteoglycan

Aggrecan

Ser Xyl Galactosamine6-sulphatase

Gal Gal

Chondroitin6-sulphate

GalNAc S

H2C O S O OH

Morquio A

GlcUA

-

COO O OH

n

H2C O S O OH OH

O

O β1-3

NH C O

OH

β1-4

CH3

NH C O CH3

Aggrecan Keratan sulphate

GlcNAc-6-sulphatase Sanfilippo D

Ser GalNAc

β-glucuronidase Sly

β−hexosamidase

Gal NANA

Gal S GlcNAc

β-galactosidase Morquio B

H2C O S O OH

n

H2C OH O OH OH OH

H C OH OH2 O

H2C O S O OH O NH

O β1-3

OH

O β1-4

C O CH3

NH C O CH3

N-acetylglucosamidase Sanfilippo B

525

28.4.2

Biochemistry and Genetics

Figure 28.7.: Spingolipidoses are caused by failure to break down sphingolipids and ceramides in the lysosome Sphingosine

(C18)

C OH

C fatty acid

O

Sphingolipid

H2C OH H HC OH HN HC OH OC O CH3 H H HO H H

H N C

Ceramide

H P Choline P Ethanolamine

head group

NANA

COO

X=

Sphingomyelin

C O X

GM1

Name

Glucosylcerebroside

Glc

(in non-neural tissue)

Galactocerebroside

Gal

(in neural tissue)

Globosides

several neutral sugars (Glc, Gal, GalNAc)

Gangliosides

complex carbohydrates: GM: single NANA GD: two NANA GT: three NANA GQ: four NANA

Ceramide Glc Gal GalNAc Gal

-

β-galactosidase

Gal

OH N-acetylneuraminic acid (NANA)

GM1 gangliosidosis

NANA GM2

Ceramide Glc Gal GalNAc Hexosaminidase A

GalNAc

Tay-Sachs disease

NANA GM3

ganglioside neuraminidase

Gal

Ceramide Glc Gal β-galactosidase

Gal

Gal Cerebroside

Gal Gal Glc Ceramide

Hexosaminidase A + B Sandhoff´s disease

NANA Globoside

GalNAc

Ceramide Glc Gal

GalNAc

Globos

Gal Glc Ceramide

α-galactosidase A Fabry´s disease

Ceramide Glc Glucocerebrosidase

Ceraminidase Farber´s disease

Sphingosine

Gaucher´s disease

Glc

Choline

Ceramide Gal Gal-cerebroside-

fatty acid

β-galactosidase Krabbe´s disease

Cerebroside

Ceramide Gal 2-

SO4

Sulphatide

526

sphingomyelinase Niemann-Pick disease

Ceramide Gal

S

cerebroside sulphatase metachromatic leucodystrophy

Phosphocholine

P Ceramide

Sphing

28.4.2

co ar s he e fa pa ce co tos rn ple e dy al nom so clo eg m stos udi aly en is ng t be al r mu ha et lti p sh vio ard lex or ral ati t bo st pro on ny atu bl he dy re ems ar sp t he va lasi ar lv a in e d g lo isea ss se

Mucopolysaccharidoses and sphingolipidoses

Disease Hunter Hurler-Scheie Maroteaux-Lamy Sly Sanfilippo A Sanfilippo B Sanfilippo C Sanfilippo D Morquio A Morquio B

 

 





  

 

       

 



   

   



 





 

GM1 -gangliosidosis Tay-Sachs Gaucher´s Krabbe metachrom. leukodystr. Sandhoff´s Fabry´s Niemann-Pick Farber´s

m

Disease

en t pa al r ra et bl lysi ard at in s io d n ch nes er s r se y-r iz ed u we res ma cu ak la he mu pa sc bo tos le ne ple sk de nom in fic e g i ki ras enc aly dn h y jo ey + in t h sk pa ear in in t fa ilu A nod cr re u o p le lip ar s id est C -lad hes or e ia n n A ea MΦ ng + io le ke ns ra to clo m ud a in g

Table 28.1.: Problems in mucopolysaccharidoses. Dysostosis multiplex: thickened skull and ribs, thick and short long bones, vertebral abnormalities

      











  

  



















 









  

 





Table 28.2.: Signs in sphingolipidoses. These diseases are usually fatal at an early age. Treatment: glycosylation inhibitors like nojirimycin, enzyme replacement therapy.

527



28.5.1

Biochemistry and Genetics

28.5. Mitochondria Mitochondria are required for energy (Krebs-cycle, oxidative phosphorylation) and fatty acid metabolism. Part of the urea cycle and heme synthesis also occur in mitochondria. Most mitochondrial enzymes encoded in nucleus → autosomal recessive inheritance. However, the mitochondrial genome contains 37 genes: • the genes for 13 subunits of OxPhos enzymes • 2 rRNAs and 22 tRNAs Mutations in those genes give maternal inheritance, as only very few (if any) of the mitochondria of the spermatozoon enter the egg during fertilization. Homoplasmy/heteroplasmy and replicative segregation affect the distribution of defect and normal mitochondria in the cells of the body, thus a mitochondrially inherited disease may break out at any time during life, and in any organ. Organs with heavy respiratory activity are however most likely to be affected: muscle, nervous system.

28.5.1. Pyruvate metabolism Pyruvate is transported into the mitochondria and then metabolized by either of two enzymes: Pyruvate dehydrogenase to enter the citric acid cycle Pyruvate carboxylase to enter gluconeogenesis Failure of either enzyme can lead to pyruvic and lactic acidosis, with significant sequelae for the patient. Pyruvate dehydrogenase Pyruvate dehydrogenase is a complex of 3 enzymes, a defect in any of these causes serious problems: lactic and pyruvic acidemia, lethargy, poor feeding, tachypnea, developmental delay, seizures, spasticity, ataxia, and sudden death. The disease may present as Leigh syndrome (vide infra). Defects in E1 α-subunit are the most frequent. This subunit is encoded on the X-chromosome, but heterozygous ~ may be affected due to X-inactivation. E1 α is inactivated by PDHkinase and activated by PDH-phosphatase. Phosphatase deficiency presents similar to E1 deficiency. The kinase is inhibited by dichloroacetate, which may be used to get as much activity as possible out of any remaining enzyme. Further management include a low carb (“ketogenic”) diet and high thiamin.

528

28.5.1

Pyruvate metabolism

Figure 28.8.: Metabolism of pyruvate. COO

CO2 GDP

-

C O P CH2 PEP

ADP

GTP PEPCK

PK

ATP COO

-

COO

C O CH2 COO

-

NADH + +H

NAD

+

COO

C O -

CH3

Oxaloacetate

-

HC OH CH3

LDH

Pyruvate

Lactate

Cytosol Mitochondrium

Pi ADP COO

-

C O CH2 COO

-

CO2 ATP

HS CoA COO

Pyruvate carboxylase

-

C O CH3 Pyruvate

-

HCO3

S CoA PDH

C O CH3 Acetyl-CoA

Oxaloacetate

529

28.5.1

Biochemistry and Genetics

Figure 28.9.: Pyruvate dehydrogenase is a complex of 3 enzymes. Mechanism of pyruvate decarboxylase complex:

E1

T H

O

+

O

C C

CH3

+

E1

H2O

O

H T C CH3

+

E2

E1 S

OH

S

SH

+

CoA-SH

+

T H

S

S-acetyl-dihydroliponamide

E2

+

H3 C C S

SH SH

E3 FAD

E2

SH SH

S

S

Liponamide

E3

530

FADH2

+

NAD+

E3

FAD

+

CoA

O

Acetyl-CoA

Dihydroliponamide

+

SH

H3 C C O

H 3C C O

E2

HCO3

E2

S

liponamide on acetyl transferase

E2

+

OH activated acetaldehyde

Thiamine-PP on decarboxylase

E1

H T C CH3

+

E3

FADH2

reduced dihydroliponamide dehydrogenase NADH

+

H+

Pyruvate metabolism

28.5.1

Defects in E3 (chromosome 7) also affects α-ketoglutarate and branched chain amino dehydrogenase, they present clinically as maple syrup urine disease with lactic and pyruvic acidemia. Pyruvate carboxylase is encoded on chromosome 11q12-q13. Three types of PC-deficiency have been described: North America (group A) lactic, pyruvate + alanine acidemia. Severe mental, psychomotor and developmental retardation. Inactive enzyme produced, amino acid substitutions. Treatment with thiamine, lipoate + dichloroacetate. France, UK (group B) respiratory distress, increased serum lactate, ammonia, citrulline, proline and lysine; intracellular redox disturbance: cytosolic compartment more reduced and the mitochondrial compartment more oxidized. Usually do not survive beyond 3 months of age. Cystic periventricular leukomalacia on cerebral ultrasound at birth. No enzyme protein produced (nonsense-mutation). ‘Benign’ type (Group C) preservation of motor and mental abilities, episodes of metabolic acidosis with elevated lactate, pyruvate, alanine, β-hydroxybutyrate, acetoacetate, lysine, and proline, managed by rehydration and bicarbonate. PC activity a few % of normal Oxidative phosphorylation is performed by complexes I–V in the inner mitochondrial membrane. Mutations in any of these complexes may cause Leigh syndrome: • Frequency: I 33 %, II 4 %, III 7 %, IV 28 %, I + IV in 28 % of cases • Early-onset progressive neurodegeneration with focal, bilateral lesions in CNS (brainstem, thalamus, basal ganglia, cerebellum, spinal cord): demyelination, gliosis, necrosis, spongiosis, or capillary proliferation. • Clinical symptoms depend on which areas of the central nervous system are involved: feeding difficulties, tachypnea, lactic acidosis, truncal hypotonia, growth retardation, cardiomyopathy, encephalopathy, myoclonic epilepsy (cave: valproate sensitivity), liver failure • inheritance mitochondrial or autosomal recessive Another disease caused by mutations in in mtDNA for complex I, III, and IV of oxidative phosphorylation is Leber hereditary optic neuropathy with acute or subacute central vision loss leading to central scotoma (blind-spot) or blindness in mid-age. Neurologic manifestations may be seen in some cases. Alcohol or tobacco abusus accelerate disease progression resulting in tobacco-alcohol amblyopia.

531

28.5.2

Biochemistry and Genetics

S CoA O S CoA

+

S CoA

Acyl-CoA (-2 C)

O

CoA-SH Thiolase

β-ketoacyl-CoA

O

NADH + H+ Hydroxyacyl-CoA dehydrogenase

NAD

L-β-hydroxyacyl-CoA

Enoyl-CoA hydratase

H2O

+

OH

O

O

S CoA

S CoA trans-∆2-enoyl-CoA

Acyl-CoA dehydrogenase

Acyl-CoA

FAD

Carnitine

CoA SH Carnitine acyl transferase II

Acyl-carnitine

FADH2

O

O

S CoA

Carnitine O

Acetyl-CoA

Figure 28.10.: β-oxidation of fatty acids.

Ragged red fibres are lumps of aggregated mitochondria in muscle biopsies which stain red with Gomori’s trichrome. They are found in myopathies caused by either of two mutations: succinate dehydrogenase subunit A (SDHA) on chr. 5p highly variable phenotype: encephalocardiomyopathy with leukodystrophy, dementia, myoclonic seizures, pigmentary retinopathy, ataxia, cardiac conduction defects, short stature, generalized muscle weakness and wasting with easy fatigability. Muscle biopsy: excessive lipid droplets in muscle fibers, mitochondria with abnormal structure and paracrystalline inclusions mtDNA for tRNALys Myoclonic Epilepsy with Ragged Red Fibers (MERRF) syndrome: progressive myoclonic epilepsy, migrainous headache and vomiting, loss of vision and/or hearing, dementia, short stature, exercise intolerance, Wolff-ParkinsonWhite syndrome in EKG: circus movement of electrical signal through Kent-bundle, resulting in sudden tachycardia. Pyruvate and lactic acidosis. Disease clinically variable because of heteroplasmy. Other mitochondrial genes (e.g. tRNAHis , tRNASer ) may also be cause in about 10–20 % of cases. Management: CoQ, carnitine, anticonvulsants.

28.5.2. β-oxidation of fatty acids All steps of β-oxidation are performed by isoenzymes with overlapping specificity for fatty acids of different chain lengths: short < 6 C-atoms

532

Peroxisomes

28.6

medium 6–12 C-atoms long 12–24 C-atoms very long > 24 C-atoms (also oxidized in peroxisome) A defect in acyl-CoA dehydrogenase leads to the accumulation of acyl-CoA of respective lengths, ω-oxidation (with dicarboxylic aciduria) and Gly-conjugation increase but can not fully compensate. short chain (SCAD) generalized (infants with aciduria) or muscle only (middle aged with myopathy). Metabolic acidosis, failure to thrive, developmental delay, seizures, myopathy, but no hypoglycemia. Autosomal recessive. medium chain (MCAD) Mostly K304E, 1p31, remaining activity < 10 %. After fasting or stress (cave: infection!) potentially fatal hypoglycemia (Glc consumption ↑, gluconeogenesis ↓ because Krebs-cycle ↓), structural changes in mitochondria. Long term damage: slowed cognitive development, cerebral edema, encephalopathy, weak muscles, exercise intolerance, fatty liver. Management: regular meals, carnitine, Glc infusion in acute crisis. long chain (LCAD) 2q34-q35, Q303K. Rare. very long chain (VLCAD) 17p13.1-p11.2, various missense mutations or deletions, some respond to bezafibrate. Membrane associated protein (other AD are soluble). Cardiomyopathy, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy, or sudden death in infancy with hepatic steatosis. Mitochondrial tri-functional protein (TFP) is a complex containing long-chain enoylCoA hydratase, hydroxyacyl-CoA dehydrogenase (LCHAD) and thiolase. Deficiency in LCHAD is a serious disease resulting in fulminant neonatal liver failure or progressive liver degeneration. Also affected are muscle, retina and peripheral nerves. ~ pregnant with LCHAD defective fetus may get acute fatty liver of pregnancy or HELLP-syndrome (hemolysis, elevated liver enzymes, low platelets). Intrauterine growth restriction of the fetus may lead to pre-term delivery.

28.6. Peroxisomes Peroxisome are formed by fission or de novo from the ER. They perform reactions that involve ROS formation, which are then destroyed by enzymes. For example the FADH2 of very long chain acyl-CoA dehydrogenase in peroxisomes transfers its hydrogens directly to oxygen, forming hydrogen peroxide which is then taken care of by catalase. The following key pathways occur in peroxisomes:

533

28.6

Biochemistry and Genetics

Plasmalogen synthesis defect in dihydroxyacetone phosphate acyltransferase in rhizomelic chondrodysplasia punctata β-oxidation of long and very long chain fa adrenoleukodystrophy because of transport defect phytanic acid oxidase infantile Refsum disease as phytol (from chlorophyll) can not be digested bile synthesis shortening of side chain by β-oxidation degradation of pipecolic acid metabolite of a minor Lys-breakdown pathway ABCD proteins are ABC-type half-transporters, that form homo- or hetero-dimers. They transport long and very long chain fatty acids into peroxisome. A defect in ABCD1 leads to adrenoleucodystrophy, inheritance is usually X-linked, with a milder form (adrenomyeloneuropathy) affecting some heterozygous ~. Some rare autosomal forms have been described (defects in the enzymes of peroxisomal β-oxidation). Failure to metabolize VLCFA leads to accumulation in brain + adrenals → demyelinization of white matter → death in adolescence. Patients present with loss of previously acquired neurologic abilities, seizures, ataxia, loss of vision and hearing and Addison’s disease. Lorenzo’s oil is probably the only film ever made in Hollywood about fatty acid metabolism. The mixture containing glycerol esters of erucic C22:1 ω9 and oleic acid C18:1 ω9 normalizes serum fa profile, may slow down disease progression in asymptomatic patients (jury is still out on that one) but does not change final outcome. Malformation of peroxisomes leads to Zellweger syndrome (cerebrohepatorenal syndrome), a severe disease usually fatal during the first half year of life. Signs include facial features flattened facies, large anterior fontanelle, widely split sutures, and broad nasal bridge polycystic kidneys with adequate functional renal parenchyma liver intrahepatic biliary dysgenesis, liver cysts, hepatomegaly, jaundice CNS generalized hypotonia with absent Moro (startling) reflex, sudanophilic leukodystrophy, mental retardation, seizures, tapetoretinal degeneration, sensorineural hearing loss joints and bones chondral calcification markers high serum iron + copper and high iron binding capacity, elevated pipecolic acid in serum and cerebrospinal fluid Peroxisomal proteins are nuclear encoded and translated on free ribosomes. The function of proteins involved in peroxisomal enzyme import is still a matter of active research, the following list is provisional:

534

Biotransformation

28.7.1

PEX3, PEX16, and PEX19 no peroxisomal membranes are formed without these proteins PXR1 (PEX5) encodes a receptor that recognizes proteins containing peroxisomal targeting sequence 1 (PTS1), defined by the carboxy terminal consensus sequence serinelysine-leucine (SKL) PEX7 encodes the PTS2 receptor and recognizes proteins with an N-terminal motif present in fewer matrix proteins. Mutations in PEX7 are associated with the clinically distinct disorder rhizomelic chondrodysplasia punctata (RCDP) PEX14, PEX17, and PEX13 docking of the PTS1 and PTS2 receptors and their associated proteins PEX10, PEX12, and PEX2 part of the translocation apparatus for matrix proteins PEX 8 anchors the above import complexes at the lumenal aspect of the peroxisomal membrane PEX1 and PEX6 recycling of peroxisomal import receptors PEX5 and PEX7 PEX1, PEX6, PEX4 and PEX22 act late in the import pathway, perhaps after the translocation process For reasons poorly understood patients may show Mosaicism: type 1 disparity between serum markers and cellular results from the same individual. In some cases, cultured cells are immunohistochemically normal at 37 °C, but show defective peroxisomes at 40 °C. type 2 disparity in matrix protein import into peroxisomes in adjacent cells from the same individual. Often corresponds to milder phenotype.

28.7. Endoplasmic reticulum The ER is required for synthesis of membrane proteins (rough ER) and for lipid synthesis, drug metabolism and sterol synthesis (smooth ER).

28.7.1. Biotransformation Our food contains many substances that plants synthesize to protect them from parasitism. Some such secondary metabolites are useful to us (e.g. as antioxidants), but many are toxic or carcinogenic and have to be removed. Pharmaceuticals are often metabolized by the same pathways. Together such “foreign” chemicals are called xenobiotics.

535

Freq. %

Exons

Gene (kb)

cDNA (kb)

Protein (aa)

Table 28.3.: Affected genes in Zellweger syndrome. The frequencies were found in a recent study on 198 patients. Patients

1283 326 + 345 359 403 377 346 299 305 373 602 + 639 980 626

Protein Name

3.9 1 1.1 1.2 1.1 1 0.9 0.9 1.1 1.8 2.4 1.5

Chromosome

41.5 7.8 3.8 31.2 135.5 8.4 5.7 10.7 39 18.9 15.1 7.4

OMIM

24 6 3 4 9 11 8 5 12 15 17 4

Gene Symbol

68.0 4.6 4.1 1.0 0.5 0.5 0.5 6.6 1.5 1.5 10.7 1.0

Peroxisome biogenesis factor 1 Peroxisome assembly protein 10 Peroxisome assembly protein 12 Peroxisomal membrane protein 13 Peroxisomal membrane protein 14 Peroxisomal membrane protein 16 Peroxisomal biogenesis factor 19 Peroxisome assembly protein 26 Peroxisomal biogenesis factor 3 Peroxisomal targeting signal 1 receptor Peroxisome assembly factor 2 Peroxisome membrane protein 3

134 9 8 2 1 1 1 13 3 3 21 2

7q21-q22 1p36.32 17q12 2p15 1p36.2 11p12-p11.2 1q22 22q11.2 6q23-q24 2p13.3 6p21.1 8q21.1

100.0

602136 602859 601758 601789 601791 603360 600279 608666 603164 600414 601498 170993

198

PEX1 PEX10 PEX12 PEX13 PEX14 PEX16 PEX19 PEX26 PEX3 PEX5 PEX6 PXMP3 (PEX2) Total

536

Biochemistry and Genetics

28.7.1

28.7.1

Biotransformation

Figure 28.11.: Phase I metabolism of xenobiotics makes the compounds more reactive and more polar, hence water-soluble. Phase I: Oxidation X H + O2 + NADPH + H

+

+ X OH + NADP + H2O

Cytochrome P-450 dependent monooxygenase

During phase I metabolism, such compounds are made more reactive and more polar by introduction of oxygen atoms into their structure. During phase II solubility is increased by conjugating polar residues (e.g. sugars, amino acids, glutathione, mercapturic acid) to them. Phase I and II xenobiotic metabolism Phase I drug metabolism makes compounds more reactive and more polar, hence watersoluble, by introducing oxygen into their structure. This is performed by a group of enzymes collectively known as cytochrome P450 (CYP450). These enzymes are monooxygenases (“mixed functional oxygenase”) which introduce one oxygen atom of an O2 molecule into the compound, the remaining atom is reduced to water with NAD(P)H. CYP450 is located on the smooth ER of liver, lung, intestine and kidney. Apart from drug metabolism it is required for ω-oxidation, fatty acid CoA desaturase, synthesis of cholesterol, steroid hormones and leucotriens and for the degradation of heme. CYP450 absorbs light of 450 nm after reaction with CO (Soret-Band), which explains their name. They can use either NADH or NADPH as co-substrate. Humans have 57 CYP450-genes (+ 5 pseudo-genes), with broad and overlapping substrate specificity.

537

28.7.1

Biochemistry and Genetics

Figure 28.12.: Reaction mechanism of CYP450. The heme-Fe and the O2 are reduced by electrons from NAD(P)H oxidation to a very reactive state (Fe+ does not even exist as isolated ions!), that can attack the substrate. Note that both binding of substrates and release of product are ordered. NAD(P)H:Cyp450 reductase (FMN + FAD-dependent)

Cytochrome P-450 (CYP450)

O2

E Fe

3+

E Fe

e 3+

H X

E Fe

2+

(

E Fe H X O2 3+

E Fe H X o O2

+

H + 2e

+

2+

-

X H

+

NAD(P) +

NAD(P)H

) ( e

-

E Fe H X O2 2+

E Fe H X oO2

H X 2+

E Fe H X CO CO absorbs light at 450 nm

538

)

H2O

+

2 H

3+

E Fe HO X H2O

HO X 3+

E Fe HO X E Fe

3+

28.7.1

Biotransformation

Figure 28.13.: In some cases activation of compounds by CYP450 makes them carcinogenic by producing highly reactive epoxides, that can introduce point mutations into DNA by reacting with G-residues. H2O, 2 NADP+

2 O2, 2 H+, 2 NADPH

O

10 9

CYP450

8

HO

7

OH (+)-Benzpyrene-7,8-diol-9,10-epoxide (causes lung cancer)

Benzpyrene (in tobacco smoke)

O

O

O

O2, H+ NADPH

O

H2O NADP+

O

O O

O

CYP450

O

O

O

H2N

H N

HN N

N R

Guanine

O

Aflatoxin B1-2,3-epoxide (causes liver cancer and kwashiorkor)

Aflatoxin B1 (in moldy food)

+

O C C

+

H

O

O +

N C C

HN spontaneous

H2N

N

N R

Point mutation

539

28.7.1

Biochemistry and Genetics

Figure 28.14.: Some harmless chemicals are activated by CYP450 into powerful poisons or highly active pharmaceuticals. Can you think of a reason to give codeine instead of morphine, if codeine needs to be activated to morphine before it has any effect? O

O

N

+

O

O2, H+ NADPH

O H2O + N O NADP+

O

GSSG, H2S

2 GSH

N

+

O

C O H2

P

O O

S O

H3C C O P O C CH3 H2 H2 O

C CH3 H2

O2, H+ NADPH

H2O NADP+

HO C O H2 O

H N CH3

HO

inactivated enzyme

Paraoxon (E600)

Parathion (Thiophos, E605)

O

CYP450

HO H2C O O

H

H

N CH3 HO

N CH3 HO Morphine

3-methoxymorphine (Codeine)

540

AChE

O

O

H3C O

Ser

OH

H3C C O P O C CH3 H2 H2 O

O H3C

O

AChE Ser OH

CYP450

H3C C O P O C CH3 H2 H2 S

+

N

28.7.1

Biotransformation

Figure 28.15.: During phase II of xenobiotic metabolism the activated compounds resulting from phase I are conjugated to polar or charged residues to increase water solubility, so they may be excreted by the kidney. Phase II: Conjugation -

-

COO

COO O

O UDP

OH

O X

O

+

X OH

+

OH

OH

UDP

OH OH

OH glucuronic acid conjugate

UDP-glucuronic acid

O O H2 O S O P O C O

O

Adenine

O

+

O X OH

3-Phospho-AMP

+

X O S O O

O

OH

O P O O Phosphoadenosine phosphosulfate (PAPS) ATP CoA-SH

Sulfate ester

AMP PPi

Gly

O

-

X COO

CoA-SH

O -

X C N C COO H H2

X C S CoA

Glycine conjugate Gln

O C NH2 O

CoA-SH

(CH2)2 -

X C N C COO H H Glutamine conjugate COOH

HCl

H2N CH

X Cl

O

CH2

+

O

CH2 O

C

N H

C CH

N H

CH2 SH

C

OH

COOH

Glu Gly

H2N CH

CH2

O

CH2

O

CH2 O

C

N H

N H

C CH

C

O

OH

H3N

CH2

(γ-Glu-Cys-Gly)

O C CH CH2 S

CH2

X

S

Glutathione

+

Cysteine S-conjugate

X

Glutathione S-conjugate Acetyl-CoA O H3C

C

O N H

O C CH CH2

CoA-SH

S X

N-acetyl-cysteinyl derivative (a mercapturic acid)

541

28.8

Biochemistry and Genetics

Pharmacogenetics Humans vary in their • ability to metabolize and excrete drugs: – organ function (liver, kidney) – drug metabolizing enzymes • affinity of drug target (receptors, enzymes) Therefore the same dose of the same drug will have different effects in different patients: • intended effects: response rate 25–75 % • side effects: – severe in about 15 % of US approved drugs even when properly administered – 2 × 106 people hospitalized per year for drug side effects – leading to 1 × 105 deaths per year → No one-size-fits-all medicine! Look out for Target selection Many diseases caused by problems in several different proteins. Pharmaceutical against target A may not work if defect is in B! Example: Cancer Heightened or reduced target affinity Increased sensitivity to side effects e.g. G6PDH deficiency Rate of transformation e.g., CYP450 polymorphisms may affect both activation and inactivation of drug Drug interactions Alcohol is also metabolized by CYP450 (microsomal ethanol oxidizing system, MEOS)→ induction → interference with barbiturate elimination. Alcohol induces the synthesis of CYP450 in the liver, but also inhibits their activity acutely. Barbiturates, for example, work in the intoxicated but not the sober alcoholic. Important in anesthesia!

542

Objectives

28.8

28.8. Objectives After completion of this course unit students should be able to • name the biochemical pathways occurring in the various organelles of a cell • discuss defects, pathomechanism, symptoms management and probable outcome of inherited diseases of metabolism • describe how xenobiotics are modified by our metabolism and what the beneficial and adverse consequences of these modifications may be • explain, using examples, how drug-patient and drug-drug interactions require individualized medicine

543

Part VI.

Semester two, Mini III

29. Advanced DNA technology 29.1. Germline Gene Manipulations (analysis of gene function) Genes can be introduced into the germline, and normal genes can be artificially disrupted by gene targeting. These methods are used to create transgenic mice and knockout mice. • These animals provide models for human diseases, and the abnormalities of knockout mice (if any) reveal the functions of the knocked-out gene. • Transgenic animals are created to secrete valuable therapeutic proteins in their milk or supply human-friendly organs for transplantation. DNA constructs for producing transgenic mice should be devoid of viral sequences and bacterial plasmid sequences, but they should carry flanking sequences that include the promoter region which you wish to use to express the gene and normally, at least one intron is needed for satisfactory expression. Techniques: 1. The gene construct is micro-injected into the male pronucleus of the zygote, or the zygote is bathed in the DNA while an electrical pulse is applied to make the membrane permeable (electroporation). The zygote is grown into an embryo, and the embryo is implanted into a pseudo-pregnant female mouse. 2. The resulting mice are PCR analyzed to demonstrate insertion of the gene construct, and the mouse that carry gene insertions are analyzed to firstly demonstrate expression of the gene and secondly to look for phenotypic effects. Problems with the above described method are large, and include the following: • Insertional mutagenesis: the insert may have disrupted an important function in the recipient. • Variable copy number of insertion: it is impossible to control how many copies are inserted into a given mouse.

547

29.1

Biochemistry and Genetics

• Lack of expression: an inserted construct may have landed in a region of the genome that is inactive (for example in heterochromatin) in the tissue of interest. However, even insertion in a permissive location will not always result in expression, because if a large copy number is inserted, the cell is likely to activate the protection against virus which inactivate tandem repeats of DNA. • With this mechanism, it is impossible to mimic loss of function mutations, as the only outcome possible is addition of gene sequences, not replacement of gene sequences. For these reasons, knock-in and knock-out methods are the currently preferred methods in research labs. DNA constructs aimed at this use generally have a central area where a difference is found relative to the native mouse sequence, flanked by long regions of perfect match to the mouse sequences. These constructs can be incorporated into the genome by double homologous recombination, one recombination event in each of the flanking regions ensuring insertion of a single copy of the experimental, central DNA construct.

For knock-out: • The central area of the construct will lead to replacement of at least one exon of the gene with “junk” DNA, and the central area also will have a copy of a gene conferring resistance to the drug neomycin. • The DNA construct is electroporated into cultured embryonic stem (ES) cells. These cells which are purified from an early embryo should be omnipotent, in other words can become part of any tissue in the mouse including the Germline. • ES cells are grown in the presence of neomycin to select for cells that have taken up the DNA. Each colony of cells is thereafter analyzed to ensure that the construct has been inserted in the appropriate position of the genome and with the wanted final structure. • Each colony of ES cells that have passed the test is inserted into the cavity of a blastocyst, which is thereafter inserted into a pseudo-pregnant mouse. The mice produced will therefore be chimeras, containing cells that originate in the ES cells as well as cells from the blastocyst. • Offspring mice are bred to produce pure mice originating from the ES cells. These mice will be heterozygotes for the introduced mutation and will be analyzed for possible phenotypic changes. • The mice will be used in brother-sister matings to produce homozygote mutant mice, which again will be used for phenotypic analysis.

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For knock-in: This is a further development of the knock-out mechanism, where the main difference is that the central area of the DNA construct carries something that is not junk but instead is of interest. This could for example be the ∆F508 mutation of the cystic fibrosis gene from a human CF patient inserted into the CF gene of a mouse to allow further analysis of the mechanism by which the mutation alters the phenotype, or to allow test of prospective drugs. The above described germline gene manipulations all involve mice. It is widely recognized that germline gene therapy in humans should be avoided because somatic gene therapy carry fewer risks and are easier to reverse should the need arise. In the far future, one could consider the introduction of useful genes to diversify the human gene pool, for example genes for vitamin C synthesis or cobalamine synthesis, or additional copies of tumor suppressor genes to reduce the cancer risk.

29.1.1. Cre/LoxP system for recombination The neomycin resistance gene is employed in both knock-out and knock-in methods. Theoretically, this could result in phenotypic effects of its own, either directly due to the protein produced or due to transcription from the neomycin promoter continuing into neighboring genes. Such considerations have led to use of the Cre/LoxP (or the similar FRT/Flip system) to take out unwanted parts of the inserted DNA: • LoxP is a DNA sequence that is included twice in the construct, flanking the region that should be deleted. • Cre is a recombinase that recognizes the specific sequence and catalyzes a reaction whereby the area between the two DNA elements comes out as a circular DNA molecule. One copy of the LoxP element is left behind, and one copy is included in the excised circular DNA. The usual setup is that the LoxP elements are inserted so that they flank the neomycin resistance gene, while the Cre recombinase is found in a commercially available mouse. When a promising knock-out/knock-in mouse has been identified, it will be bred with the Cre-containing mouse and the offspring should loose the neomycin gene. Conditional knock-out: Another interesting application of the Cre/LoxP system is based on the availability of mice which harbor a Cre gene that is only expressed in a defined tissue or at a regulated time during development. In this setup, the initial knock in replaces an exon in the gene of interest with the same exon but now flanked with LoxP-elements. Breeding the resulting mouse with the tissue-specific or temporally-specific Cre mouse leads to loss of the exon but only in the tissue of interest, such that the effects of a mutation

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in the liver can be studied independently of the effects of the same mutation in another tissue.

29.2. RNAi (inhibitory RNA, Knock-down) Some diseases are caused by the expression of undesirable genes; viral genes in virus diseases, oncogenes in cancer, and regulatory genes in some autoimmune diseases. RNAi methods are aimed at reducing expression of a specific gene in a cell, either a cell culture for analytical purposes (what does the gene do) or as an experimental drug. The mechanism of action is a little different, but this is an extension of the methods known as anti-sense technology. RNAi is using a mechanism in the cell that was developed as a defense against double-stranded RNA virus. It seems that a double stranded RNA molecule will first be cut into shorter pieces by a protein called Dicer, that the resultant shorter fragment will be attached to a complex known as RISC, and that it in that complex will be found in single stranded form. Another single-stranded RNA molecule (e.g., an mRNA) can hybridize with the RNA on the RISC complex; it will then be cut into shorter pieces and the hybridizing part will be released while the RISC complex including the original single stranded RNA can be recycled. Adding an RNA molecule that is complementary to an existing mRNA in a cell can therefore produce the double stranded molecule that initiates the RNAi process, and effectively eliminate translation of a given protein in the cell.

29.3. Microarray Technology (DNA Chips) In traditional probing (dot blotting, Southern blotting), the probe is applied to the immobilized target DNA. To test the target DNA for all theoretically possible mutations (“mutation scanning”), a large number of probes would have to be applied successively; this would therefore take prohibitively long time. Using many different probes immobilized in a grid pattern on a solid support and simultaneously hybridize all of these with a chopped up version of the target DNA is the principle in microarray technologies. The target DNA will be labeled with fluorescent dye, left to interact with the probes for hours to days, nonbound target DNA will be washed away, and the amount of hybridization will be quantified by a laser-equipped reader. Gene Chips: Gene chips are used for gene scanning, or the simultaneous analysis of many mutations or polymorphisms: in this application, the probes each consist of one allelespecific oligo (ASO - normally about 15 bases long with the central position differing between the mutated and the normal form of the gene). Since up to 100 000 probes fit on a

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single chip, all scanning and recording has to be computerized. Applications of gene chips, or microarrays, include: • Mutation scanning of individual genes in patients, to figure out whether a clinical problem is caused by a defect in a specified gene. • The genome-wide genotyping of polymorphisms, especially single-nucleotide polymorphism (SNPs), in the context of association studies aimed at finding disease causing mutations (more on association studies in the section on Multifactorial Disease). There are a few million SNPs in our genome, but only a few of them (several thousand?) are medically important. Also, sequence differences between related species (e.g. human and chimp) can be detected with gene chips. In summary, rather than screening for one mutation at a time, people can be screened for thousands of single-gene disorders, susceptibility genes for multifactorial diseases, and normal polymorphisms, all in the same procedure. DNA chips are expected to become the standard method for predictive genetic screening, for example in prenatal or preimplantation diagnosis. cDNA Chips: cDNA chips are used to study patterns of gene expression. The immobilized probes are not ASOs but are either cDNAs or longer oligonucleotides corresponding to a unique portion of a cDNA and they are not used to analyze genes, but mRNA. The mRNA is extracted from the cells or tissue, usually simultaneously reverse transcribed into a cDNA and labeled with a fluorescent group, and then hybridized to the cDNA chip. Since each cDNA on the chip corresponds to one gene, you can quantify which genes are expressed and how much. Most often, the experimental design calls for competitive hybridization with samples coming from two different sources, e.g., the pattern of gene expression in a tumor can be compared with gene expression in the normal cells from which the tumor originated. This application is expected to soon be used in the clinic, as the expression pattern in the tumor has prognostic value for the patient and has value for the oncologist in picking the drug cocktail most likely to be effective on this particular tumor.

29.4. Somatic Gene Therapy Many genetic diseases are caused by inactivating (“loss-of-function”) mutations. In theory, these diseases can be treated by introducing an active, functional gene into those somatic cells where it is needed. Gain-of-function mutation can theoretically be treated with a DNA molecule that produces an mRNA which initiates an RNAi reaction and thereby hinders expression of the damaging protein. Problems:

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1. Large DNA molecules are not easily taken up by cells. 2. Even if it enters the cell, the gene may be degraded by nucleases. 3. Exogenous genes rarely become integrated into the cellular genome, although this is theoretically possible by homologous recombination. 4. The regulation of gene expression is problematic for artificially introduced genes. Therefore, gene therapy is promising only if tightly-regulated gene expression is not required. 5. The targeting of a gene to the relevant tissue is difficult. 6. Somatic gene therapy does not affect the Germline; the patient can still transmit the defective gene to his children. Many consider this an advantage of somatic gene therapy! In the United States, some gene therapy trial participants have died as a result of the therapy given. This has lead to a reconsideration of the safeguards necessary before an experimental gene therapy can be tested in the treatment of human disease. Integrating the therapeutic gene into viral vectors is probably the most active research field in gene therapy. Retroviral vectors are attractive because they insert the gene directly into the cellular genome. They contain retroviral long terminal repeats, but the gag, pol, and env genes are replaced by the transferred gene, all nicely wrapped in a retroviral capsid and envelope. The reverse transcriptase + integrase in the virus particle effect reverse transcription and integration into the genome. Problems: • Only low titers can be produced. • Only dividing cells can be transfected by currently used retroviral vectors. Lentiviral vectors (related to the AIDS virus) are promising because they can infect nondividing cells. Retroviral vectors and Adenoassociated Virus (AAV) suffer from limitation in size the DNA that can be engineered into the virus, while Adenovirus can hold much more DNA. Adenovirus can be produced in high titers and infect nondividing cells, but they rarely integrate into the host cell genome. They also have the potential of damaging the infected cells. Adenoassociated Virus (AAV) integrate into the genome of the host cell and are some of the most promising viral vectors for therapy involving small genes. In some contexts, repeated treatment would be necessary, and antibody produced against the viral vector becomes a big problem. Physical methods of gene delivery (liposomes, receptor-mediated endocytosis) could potentially be helpful in these circumstances. Gene therapies are still experimental. They are not only promising for the treatment of genetic diseases, but also for some other diseases, for example, bringing genes for antiinflammatory proteins into the joints of arthritis patients. Many gene therapy protocols

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are ex vivo techniques, and only a small proportion (Usually less than 1 %) of the cells are stably transfected. Ex vivo means that for example bone marrow cells are aspirated, grown in cell culture and transfected with the therapeutic gene; the gene insert is analyzed before the cells are infused back into the patient. A similar process can be performed with liver cells, but obviously not with muscle or nerve cells. A single gene therapy protocol have moved beyond the experimental into the approved protocols; the Chinese government has approved to inject a type of solid tumors with a DNA construct containing the P53 protein. The expected outcome is to stop cell divisions due to the sensing of DNA damage, and when combined with traditional chemotherapy, to induce apoptosis in the cells that have taken up the gene construct.

29.5. Proteomics Methods Proteomics entails the study of the complete complement of proteins encoded by an organism. Separation of proteins is usually accomplished using two-dimensional (2D) gel electrophoresis. Firstly, proteins are separated by charge using isoelectric focusing (IEF); secondly, the separated proteins are then further purified by separation in a second dimension based on size differences (using SDS in the gel). The 2D gel can then be used as a template to analyze the proteome using antibody-based technology or chemical analysis. Mass spectrometry (MS) technology allows sensitive and accurate detection of proteins in 2D gels. For large proteins, fragmentation with a specific protease may be necessary before using MS, while smaller proteins can be analyzed directly. For instance, the slight mass difference in the sickle-cell hemoglobin protein can be distinguished from the normal beta-hemoglobin protein using a single spot on a 2D gel analyzed in a mass spectrometer.

29.6. Objectives • Transgenic mice: understand methods, compare and contrast for especially what is the outcome of – Traditional – Knock out – Knock in – Conditional – Realize that the Cre-LoxP system can be used to delete a part of the inserted construct in vivo

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• Knock down technology: understand the differences from and similarity to knock out • Describe the use of allele-specific oligonucleotides in a chip format to test for a persons genotype • Describe the use of longer oligonucleotides on a chip to measure expression level of a gene – Tumor as an example with the data improving diagnosis and choice of treatment • Use of proteomics methods to identify completely unknown proteins – Describe use in identification of new pathogens – And in identification errors in protein modification • Gene therapy – Describe use of several different delivery systems – The difficulty of making a system that is beyond “experimental” • Recognize existence of therapies involving inhibitory RNA

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ROSS UNIVERSITY SCHOOL OF MEDICINE

30. Population Genetics and Genetic BIOCHEMISTRY AND GENETICS II Counseling Handout 26

POPULATION GENETICS AND GENETIC COUNSELING

30.1. Genotype Frequencies

I. GENOTYPE FREQUENCIES 30.1.1. The Hardy-Weinberg equation 1. The Hardy-Weinberg equation

The Hardy-Weinberg equation describes the frequencies of genotypes for an autosomal gene with alleles. It reads: The two Hardy-Weinberg equation describes the frequencies of genotypes for 2 2pq + q 2 = 1 (30.1) an autosomal gene with two alleles.p It+reads:

A p

a q

A p

2

p

pq

a q

pq

q2

p = gene frequency of allele A q = gene frequency of allele a p2 = frequency of genotype AA 2pq = frequency of genotype Aa q2 = frequency of genotype aa

With With two alleles A andAa and in one locus, + q =p1. + q = 1. two alleles a in one plocus, Remember that gene frequency is synonymous with allele frequency.

Remember that gene frequency is synonymous with allele frequency

The Hardy-Weinberg equationequation is used toiscalculate frequencies all genotypesof when The Hardy-Weinberg used tothe calculate theoffrequencies all the frequency of one genotype is already known. Example: You know the population incidence genotypes when the frequency of one genotype is already known. Example: of aknow recessive disease and incidence want to know carrier frequency. The want gene (allele) You the population of athe recessive disease and to knowfrequency the can befrequency. calculated from thegene disease frequency.can be calculated from the disease carrier The frequency

frequency. • For rare autosomal dominant traits, the gene frequency is about one half of the disease -frequency. For rare autosomal dominant traits, the gene frequency is about one half of the disease frequency. • -ForFor rarerare autosomal recessive traits, the genethe frequency the squareisroot the disease autosomal recessive traits, gene is frequency theofsquare frequency. root of of the disease frequency. For X-linked traits, the gene frequency is equal to the disease frequency • For X-linked traits, the gene frequency is equal to the disease frequency in males. in males. The Hardy-Weinberg equation can be used only when:

The Hardy-Weinberg equation can be used only when: a) b) c) d)

The different genotypes have equal fertility. 555 The different genotypes have equal viability. Mating is random for the trait. There are no new mutations.

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Biochemistry and Genetics

• The different genotypes have equal fertility. • The different genotypes have equal viability. • Mating is random for the trait. • There are no new mutations.

Assortative mating Mating is positively assortative when carriers of the same trait have an increased likelihood to mate with each other, and negatively assortative when they have a decreased likelihood of mating with each other. Mating tends to be positively assortative for most externally noticeable traits including height, education, beauty, deafness, obesity, religion, money, etc. Positively assortative mating increases the incidence of homozygosity for simple Mendelian traits. For quantitative traits, it favors the more extreme phenotypes.

Balanced polymorphism A locus is considered polymorphic when the frequency of the most common allele does not exceed 0.99. At least 30 % of all loci are polymorphic. Each individual is heterozygous at about 7 % of his gene loci. In most cases, the variant alleles do not cause disease. Balanced polymorphism is a genetic polymorphism stabilized by selection. The most important mechanism is heterozygote advantage, which favors the less common allele. It probably important for the maintenance of many common polymorphisms: Blood groups, HLA antigens. It also maintains certain recessive disease genes in the population, as in the case of hemoglobin S. In the absence of heterozygote advantage, polymorphisms can be maintained by random drift when there is no selection against them. Or they represent an evolutionary transition from a more “primitive” allele to a more “modern” one.

Genetic drift The random variation of gene (allele) frequencies in small isolated populations is called genetic drift. Founder effect is the randomness associated with choosing the few “founders” of a population, e.g., the few founders of one of the small genetic sects, or the survivors of a natural catastrophe. Together these effects can lead to the unusually high incidence of an otherwise rare genetic disease in a small or once-small population that is descended from only a few founders. On the flip side, these effects also often lead to reduced genetic variation in the population (genetic drift is most important for this).

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Mutation and Selection

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30.2. Inbreeding Degrees of relatedness The percentage of shared genes is: Monozygotic twins: 100 % First-degree relatives (siblings, parent, child): 50 % Second-degree relatives (half-sibs, uncle, grandparent...): 25 % Third-degree relatives (grand-grandparent, first cousin): 12.5 % Consequences of inbreeding The coefficient of inbreeding indicates the proportion of gene loci at which a person is homozygous due to inbreeding. For the child of a first-cousin marriage, the coefficient of inbreeding is 0.0625. For the child of a brother-sister mating, it is 0.25. Consequences: • Inbreeding increases the frequency of homozygosity for rare recessive allele. This effect is stronger for rare alleles than for common ones. It increases the incidence of rare recessive diseases. About one-third of the children of incestuous matings between firstdegree relatives are physically or mentally abnormal, and many of these problems can be diagnosed as recessive diseases. A different consequence of this is that for rare recessive diseases, quite a few of the patients will have parents that are (sometimes remotely) related. • Multifactorial traits may be adversely affected. This includes slightly increased early mortality and decreased IQ in children of first cousins. This inbreeding depression is caused by homozygosity for mildly deleterious mutations and the loss of heterozygote advantage.

30.3. Mutation and Selection Mutation rate Mutations are either spontaneous (“basal mutation rate”) or induced by chemicals or radiation. According to current estimates, each child is born with about 100 new mutations. Most of them are point mutations in the junk DNA and totally harmless, but perhaps 2 or 3 on average are mildly unfavorable and can contribute to multifactorial disease. About 1 in 200 children is born with a new mutation that is sufficiently serious to cause a diagnosable disease. Among disease-causing mutations, the highest mutation rate is for fragile X. Some genes with high mutation rates (neurofibromatosis, Duchenne muscular dystrophy) are very large. Mutation rates can easily be determined for autosomal

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Biochemistry and Genetics

dominant and X-linked diseases, but not for autosomal recessive. Advanced paternal age is the most important risk factor for new point mutations (but not deletions). Selection against mutations The fitness of a genotype is measured by the number of offspring produced. A fitness of zero (no offspring) is a genetic lethal. A fitness of 1 means normal fertility. Low fitness does not always imply poor health or early death. Selection often works by differential reproduction rather than differential survival — especially in countries with good medical care. Selection against autosomal recessive disease genes is very inefficient because most of the time they are hidden in unaffected heterozygotes. Inbreeding selects against bad recessive mutations because it exposes them to selection in the homozygous state. Selection and single-gene disorders • Some “harmless” traits such as color blindness and pattern baldness are very common because they do not compromise reproduction. • Genes for some serious diseases, like Huntington’s disease and the dominantly inherited forms of Alzheimer’s disease are maintained because they strike after the peak reproductive age. (Some studies show that male carriers of Huntington disease on average have more children than their male non-carrier siblings). • If the incidence of a disease is constant, then the bad alleles weeded out by selection in the previous generation must have been replaced by new mutations. As this is the case in most diseases we know of, autosomal recessive diseases must have a relatively low mutation rate. • There is an inverse relationship between the fitness of an autosomal dominant mutation and the proportion of patients having a new mutation. If achondroplastics have a fitness of 0.2 (20 %), then 80 % of them have a new mutation. • Some recessive disease genes are maintained by heterozygote advantage: sickle cell, thalassaemia and glucose-6-phosphate dehydrogenase deficiency for malaria protection, and cystic fibrosis for protection from diarrheal diseases. Also lipid storage diseases in Jews may be due to heterozygote advantage. Selection for quantitative traits Stabilizing selection is present when persons at both tail ends of the normal distribution have reduced fitness. It reduces the standard deviation for the trait without changing the mean.

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Mutation and Selection

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Disruptive selection favors extreme phenotypes over average phenotypes. It increases the standard deviation for the trait. This pattern is rare. Directional selection favors phenotypes at one end of the normal distribution over those at the other end. It changes the mean of the normal distribution. Stabilizing selection is common both for health-related traits (example: blood pressure) and for traits that are important for mate choice (example: height). Directional selection can be strong for traits like socioeconomic status, education, intelligence, and ethnic or religious background. The best-documented selection effect in modern societies is selection against female intelligence. Other examples: Catholics and protestants in Northern Ireland, black and white South Africans, Anglos and Latinos in the US. Selection for quantitative traits is the mechanism of evolution.

Selection and multifactorial disorders The risk of multifactorial diseases is influenced both by freak mutations that increase disease risk but are not serious enough to be removed speedily by selection (“genetic garbage”), and by common polymorphisms. Other considerations: • Senior citizens are not as healthy as young folks because there is no selection against those polygenic diseases that strike after the reproductive age. • Some multifactorial traits may have been adaptive in the past but lead to problems in modern societies. Genes for obesity and type II diabetes can be adaptive under conditions of limited food supply.

Medical intervention and selection Does medical intervention increase the incidence of heritable (Mendelian or multifactorial) disease? • In some multifactorial disorders, life-saving surgery increases the disease incidence in the next generation. Parents who survived pyloric stenosis or congenital heart defects thanks to surgery often produce children with the same defect. But only a small percentage of children born with these defects have an affected parent, therefore the effect is slight. • Selection against some single-gene disorders is relaxed by medical intervention. This can increase the disease incidence noticeably for some dominant and X-linked diseases, but not for recessives. • The incidence of diseases that are expressed after the reproductive age is not likely to increase through medical intervention.

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Biochemistry and Genetics

30.4. Genetic Counseling Aim Most genetic diseases are hard to treat. Therefore, extra attention should be given to disease prevention. Typical situations: • The prospective parents already have a child with a supposedly genetic disease. They want to know if there is any risk for their future children. • One of the prospective parents has a serious disease. • A relative of the parents has a possibly genetic disease. • The prospective parents are consanguineous. • One of the prospective parents is very old. The physician is expected to provide information about the nature of the disease, the risk for the disease in future children, and available options for disease prevention. Genetics clinics Genetic counseling is an integral part of medical practice. The primary care physician is, for example, expected to inform an elderly pregnant patient about the risk of Down’s syndrome and the possibility of prenatal diagnosis. And he is expected to inform the mother of a cystic fibrosis child about the recurrence risk and possibility for prenatal diagnosis. Failure to provide genetic counseling in such situations is malpractice! More complicated cases should be referred to a genetics clinic. Genetics clinics exist in many university hospitals and major medical centers. There are specialized clinics for the more common diseases, such as sickle cell and cystic fibrosis. Population risks Some relevant population risks for conditions, which may or may not have a genetic background, are: Infertile couple: 1 in 10 Pregnancy ending in spontaneous abortion: 1 in 8 Child born with serious handicap: 1 in 50 Perinatal death: 1 in 30 to 1 in 100 Death during first year of life, after first week: 1 in 50 Diagnosis Take a careful family history. Inquire about spontaneous abortions, stillbirths, infant deaths, and consanguinity. If the condition may be either genetic or environmental (examples: mental retardation, deafness), a careful evaluation of possible environmental causes is essential.

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Diagnostic Strategies

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Risk estimate Mendelian ratios can be calculated for established single-gene disorders. Otherwise you use empiric risks. Mendelian risks for an individual pregnancy are unaffected by the birth of a previous affected child, in multifactorial traits the birth of another affected child increases the empiric risk in future pregnancies. Many disorders show genetic heterogeneity or phenocopies. When differential diagnosis is not possible, an empiric risk may have to be applied to a supposedly Mendelian disorder. Example: An isolated case of deafness may be caused by autosomal recessive inheritance or by an unrecognized environmental agent. The current trend is that more and more genetic etiologies are becoming diagnosable by molecular methods.

Options Frequently, a supposedly genetic condition turns out to be non-genetic, or the recurrence risk of a multifactorial condition is so low that the prospective parents need not worry. However, if there is a substantial risk of a severe disease, there are several options: • No children. Or a lover with good genes. These are the old-fashioned recommendations. It is still a reasonable option in some cases, for example when a parent has isochromosome 21 or the mother has PKU. • Artificial insemination by donor (Nobel prize winner or Olympic gold medal winner, if available). This makes sense in cases of male infertility and when the prospective father may contribute a gene for a serious disease. Not acceptable for all couples. • Prenatal diagnosis with selective abortion of any affected fetus. This method tends to replace the others, but it is not acceptable for everyone or legal everywhere. Also, not all diseases can be diagnosed prenatally. • Preimplantation diagnosis with selective implantation of unaffected embryos. This avoids the unpopular step of pregnancy termination, but it requires in-vitro fertilization. Expensive! Remember that counseling should be non-directive, the choice belongs to the patient, not the doctor!

30.5. Diagnostic Strategies Heterozygote detection Heterozygotes can be identified for many, but not all autosomal recessive and X-linked recessive disorders. Traditionally, these tests are applied only in rel-

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atives of a patient. Disease Cystic fibrosis Sickle cell disease Thalassemias Phenylketonuria Mucopolysaccharidoses Lipid storage diseases Galactosemia Hemophilia A Duchenne muscular dystrophy G6PDH deficiency

Test Used Allele-specific probes Sickledex, dot blotting Hemoglobin electrophoresis, RBC morphology, DNA tests Phenylalanine tolerance test Enzyme activities “ “ “ “ Linked markers Serum creatine kinase, PCR/Southern blot Enzyme assay, electrophoresis

Population screening Heterozygote screening makes sense only when: • The disease is relatively common • The disease is severe enough to justify prenatal testing and pregnancy termination. • The screened population is sufficiently educated to understand what it’s all about. Good candidates for population screening are sickle cell, cystic fibrosis, Tay-Sachs and fragile X. Because most people don’t care much about genetic risks until they are expecting a child, most screening programs are aimed at pregnant women.

Newborn screening Newborn screening is used for severe but treatable conditions. It is often done for congenital hypothyroidism, PKU, other treatable metabolic diseases such as galactosemia and biotinidase deficiency, hemochromatosis, and α1 -antiprotease deficiency.

30.6. Prenatal Diagnosis 30.7. Aim Prenatal (=antenatal) diagnosis is performed when the fetus is at risk of a severe disease. The test is done as early as possible, and the pregnancy is terminated if the fetus is affected.

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Screening Tests

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30.8. Screening Tests Screening tests are noninvasive tests that are done routinely in every pregnancy. Ultrasound is part of routine prenatal care. It detects most cases of twin pregnancy and gross malformations. Can also detect e.g., Turner syndrome: should pregnancy termination be offered? There is at least 99 % risk of spontaneous abortion, but quite normal phenotype after birth (increased risk of heart defects). Maternal blood tests: Maternal serum α-fetoprotein is often elevated in neural tube defects (NTD’s) and reduced in Down syndrome. Also popular: Triple test with α-fetoprotein, human chorionic gonadotropin and estradiol. For Down and NTD’s. Suspicious cases are referred for amniocentesis.

Amniocentesis In amniocentesis, performed at week 15 or 16, a small amount of amniotic fluid is removed in a transabdominal approach, under ultrasound guidance. The fluid contains viable cells sloughed off from the fetal membranes. These cells, which have the fetal genotype, can be cultured like fibroblasts. Culturing takes 1–3 weeks. Diagnostic tests are done on the cultured cells. The risk of the procedure (fetal damage, infection, induced abortion) is minimal. Indications: Chromosome aberrations, Down syndrome in particular, are the most common indication. Pregnancies of women older than 35 a are routinely monitored by amniocentesis. Mendelian disorders are tested when both parents are carriers of an autosomal recessive disease gene, one parent has dominant disease, or the mother is a carrier for a X-linked recessive disease gene. Neural tube defects are tested in families who already had a child with a neural tube defect. The level of α-fetoprotein is determined in the amniotic fluid. In many places, α-fetoprotein is measured routinely with every amniocentesis, even if the primary indication was different (usually advanced maternal age).

Chorionic villus sampling In this procedure, a biopsy is taken transvaginally from the chorionic membrane. The risk may be slightly higher than for amniocentesis and you cannot test for neural tube defects, but • It can be done earlier, at week 8–10 • More cells are obtained. Therefore cell culturing is not always necessary. This saves another 1–3 weeks.

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Effects on disease incidence Even if the birth of children with recessive diseases is avoided by prenatal diagnosis and selective abortion, the allele frequencies will not necessarily decline because most parents keep trying until they have the desired number of children. Two thirds of these children will be carriers. This is called reproductive compensation. Ethical and legal concerns Attitudes toward prenatal diagnosis and pregnancy termination vary in different cultures. In most Western countries and ex-Communist countries, prenatal diagnosis and the abortion of fetuses with severe diseases is legal. In Western countries, the decision about pregnancy termination is made by the parents, not the doctor. Because many diseases are now avoidable, the question arises of whether parents who opt for a chronically ill child should be liable for the medical expenses. Sex-selective abortion after ultrasound or amniocentesis is now considered acceptable in the US. Only a minute proportion of pregnancy terminations worldwide are performed on diseased fetuses after prenatal diagnosis.

30.9. Assisted Reproduction Incidence of infertility Fertility problems occur in perhaps 10 % of younger couples and > 30 % of couples beyond age 35 a. 40 % are caused by male infertility due to infections (ie. Mumps), chromosomal abnormalities (most important: Klinefelter), immunological problems (sperm autoantibodies), anatomical problems, microdeletions on the Y chromosome etc. The male partner is always evaluated first because the diagnostic workup is easier in the male. The two most common causes of female infertility are ovulatory dysfunction and tubal/pelvic pathology. Advanced age is the most important and least treatable cause of female infertility! Artificial insemination by donor (AID) AID is used for intractable male infertility, and by single women. It can be used when there is a history of fetal loss due to rhesus incompatibility, and in cases of genetic diseases when the male partner may contribute a severe disease gene. The success rate depends on the age of the patient, but typically 15–20 % per cycle with fresh semen, 5 % with frozen semen. Technicalities and precautions: • Semen can be quick-frozen in the presence of an antifreeze (10 % glycerol). It keeps almost indefinitely in liquid nitrogen. Sperm banks express-mail their product in nitrogen tanks to the doctor’s office where the insemination is performed. • Married women should be inseminated only after both partners have signed a consent form. This is legally required in 20 states. • Insemination should be timed to coincide with the LH surge. Take-home kits for LH measurement are available.

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Assisted Reproduction

30.9

• Practicing homosexuals, men with multiple sexual partners, and intravenous drug users are less-than-optimal donors because of the risk of sexually transmitted disease. • Frozen semen should be quarantined for 6 months, and repeat AIDS testing of the donor performed before the batch is released. This is mandatory in most states. • Most sperm banks use students as donors, and they apply an age limit (35 or 40 a) because old sperm may have reduced sperm count or motility, and because of an increased risk of new mutations. The typical pay for donors is about US$ 80. • Most but not all sperm banks have only anonymous donors. • Donor selection includes complete physical, semen analysis, testing for transmissible diseases (AIDS, syphilis, hepatitis), educational history, and family history for Mendelian and multifactorial diseases. Carrier testing for sickle cell (Blacks), thalassaemia (Mediterraneans), Tay-Sachs (Jews) and cystic fibrosis (everyone) is increasingly used. Some sperm banks are specialized on Nobel prize winners or homosexuals (for lesbian customers). • Sperm banks have catalogs for initial donor selection, and detailed donor profiles can be obtained on request. Married couples often select donors with physical characters similar to the husband’s. The physician should advise the patient about the heritability of relevant traits. If the patient has a multifactorial disease, the use of a donor with a family history of the same disease should be avoided. • During routine insemination, semen is deposited at the entrance to the cervical canal by means of a polyethylene catheter connected to a 1 ml syringe. • Alternatively, intrauterine insemination (IUI) is performed with washed resuspended sperm. • Sperm enriched in X- or Y-bearing sperm cells is offered by many sperm banks. Separation of the cells is always very incomplete, however. In-vitro fertilization (IVF) IVF was first used in women with tubal disease, but is now also used for male factor infertility, unexplained infertility, and infertility caused by endometriosis or immunological factors. While AID is usually done in the doctor’s office (or even by the patient herself at home), IVF is done in specialized clinics. Procedure: • Hormonal stimulation is used to induce superovulation (maturation of multiple follicles). These treatments raise the gonadotropin level during the follicular phase. It can be done by exogenous human menopausal gonadotropin (HMG), human chorionic gonadotropin (HCG), recombinant FSH, or clomiphene citrate, an estrogen partial agonist/antagonist which blocks the negative feedback of estrogen on gonadotropin secretion.

565

30.9

Biochemistry and Genetics

• Follicles are retrieved at mid-cycle under ultrasound guidance. Analgesics (fentanylmidazolam) are used. Up to a dozen follicles can be harvested (if you’re lucky). • The oocytes are prepared under the dissection microscope, incubated for several hours, and then exposed to washed, resuspended sperm. • Embryos can be transferred any time between the pronuclear and blastocyst stages, but transfer is usually done at the 4–10 cell stage (48–80 h after retrieval). About 3 embryos are placed in younger women, more in older women. Implantation rates are low, but placement of too many embryos increases the risk of multiple pregnancy. • Excess embryos are cryopreserved for later use. Freezing embryos is technically easier than freezing oocytes. • The delivery/retrieval pregnancy rate is about 20 % (10–15 % per cycle). The cost is US$ 6000 to US$ 10 000 per treatment cycle. • The incidence of multiple pregnancy after IVF is 30 %. Also ectopic pregnancies are common (4–5 %). There is no increased incidence of congenital malformations. There is no significant increase of developmental abnormalities in babies obtained from frozen embryos. • IVF can be done with embryos from donated sperm and ovum (intrauterine adoption). This is an option for postmenopausal women and women with ovulatory failure, but it is rarely done because donated oocytes are in short supply. Intracytoplasmic sperm injection (ICSI) A sperm cell is injected directly into the cytoplasm of the ovum. This method can overcome problems with sperm motility or maturation, and it can be used with sperm cells obtained by epididymal puncture in patients with atresia of the vas deferens or post-vasectomy. The method seems fairly safe. Thanks to ICSI, there are now cases of truly inherited male infertility. Importance of assisted reproductive technology (ART) Between 1.5 and 2.0 % of children in the US are born through ART, about half of them by donor insemination and the other half by IVF. The procedures are not usually paid by the health insurance in the US, but they are covered, with qualifications, by the health care systems of many other countries. About one-half of all married recipients tell their child of his/her origin. Divorce rates are lower than usual in couples having an ART child, and parenting tends to be better. There may be a tendency for children of well-screened donors to be healthier than naturally conceived children, and smarter if the donor is a Nobel Prize Winner. Lesbians and career women are now a major clientele. Children of single women and lesbians conceived by ART do as well as those of married couples. Therefore there is no reason to deny treatment to these customers.

566

Objectives in Brief

30.10

Pre-implantation genetic diagnosis This is an advanced method for the diagnosis of single-gene disorders (using PCR) and chromosome aberrations (using FISH), done only in a few places. Procedure for single-gene disorders: • Grow the IVF embryos to the 8- or 16-cell stage. • Remove a single cell from each embryo. This does not interfere with embryonic development. check – there was a recent paper (in Nature IIRC) stating the opposite • Use the DNA of this single cell to diagnose the disease with PCR. The test takes about 8 hours. • Discard the bad embryos and implant the good ones. Pre-implantation diagnosis is a costly alternative to prenatal diagnosis for parents who don’t like the idea of an abortion at all. Otherwise, it makes most sense in cases where IVF is done anyway.

30.10. Objectives in Brief 1. State the criteria for validity of the Hardy-Weinberg equation 2. Use the Hardy-Weinberg equation to predict the allele frequency and the frequency of different genotypes for single gene disorders if the disease incidence is known. 3. Define the terms heterozygote advantage, genetic drift, founder effect and assortative mating. 4. Predict the effects of inbreeding and incest on the incidence of autosomal recessive and multifactorial disorders. 5. State the approximate mutation rates of genes for important Mendelian disorders, including Huntington’s Disease, Achondroplasia, Neurofibromatosis, Hemophilias A and B, and Duchenne’s Muscular Dystrophy. 6. Describe the expected effects of negative selection on allele frequencies of autosomal recessive and dominant diseases, and explain why the lack of effects makes negative eugenics unethical and scientifically wrong. 7. Define the term stabilizing selection, disruptive selection, and directional selection and give examples for each. 8. Define the aims of genetic counseling and give the typical sequence of steps.

567

30.10

Biochemistry and Genetics

9. State the most likely causes for the observed population incidences of important genetic conditions including Duchenne’s Muscular Dystrophy, Achondroplasia, Huntington’s Disease, Sickle Cell Disease, Thalassemias, Glucose-6-Phosphate Dehydrogenase Deficiency, Cystic Fibrosis, Obesity, Type II Diabetes, Alzheimer’s Disease, Osteoporosis, Red-green Color Blindness, Pattern Baldness, Homosexuality, Mild Mental Retardation, and Skin Color Differences. 10. Describe the procedure of amniocentesis, chorionic villus sampling and pre-implantation diagnosis. 11. Specify the typical situations in which prenatal diagnosis for chromosome aberrations and Mendelian disorders is indicated. 12. Compare the advantages and disadvantages of amniocentesis and chorionic villus sampling for prenatal diagnosis. 13. Provide examples of congenital or hereditary diseases for which newborn screening is currently practiced or proposed. 14. Describe the most important methods of assisted reproduction including artificial insemination, in vitro fertilization and intracytoplasmic sperm injection, and list three typical indications for each of these procedures. 15. State the ethical and legal implications of current procedures for prenatal diagnosis, pre-implantation diagnosis and pre-symptomatic diagnosis. 16. State the effect of paternal age on rate of point mutations.

568

31. Integration of Metabolism 31.1. Regulation of Enzyme Activity Mechanisms regulating key metabolic enzymes include: • Allosteric regulation, usually by metabolic substrates, intermediates, or end products, acts on many key enzymes in metabolic pathways. Also competitive inhibition by metabolites is important. Action is immediate. • Phosphorylation/dephosphorylation is regulated by hormones (glycogen phosphorylase, adipose tissue lipase, acetyl-CoA carboxylase) or metabolites (pyruvate dehydrogenase). This is the most important mechanism by which second messengers (cAMP, cGMP, diacylglycerol, Ca2+ ) induce their effects. Insulin induces the dephosphorylation of many metabolic enzymes although its initial effect is the phosphorylation of regulatory proteins. Action is on an intermediate time-frame. • Adaptive control, meaning adjustments in the synthesis or degradation of metabolic enzymes, is a long-term (day-to-day) type of control. Enzyme synthesis is often under hormonal control. Steroid and thyroid hormones affect transcription directly. Watersoluble hormones achieve the same through their second messengers which induce the phosphorylation of transcription factors and other nuclear proteins. Metabolites can also regulate gene expression.

31.1.1. Hormonal Control Hormones coordinate the activities of the metabolic pathways to adapt to the nutritional state and physiological requirements. Insulin satiety hormone: release stimulated by Glc and AA. Stimulates nutrient uptake from blood into cells and synthesis of storage compounds (glycogen, fatty acids, triglycerides, cholesterol) and inhibits the breakdown of stored nutrients, including fat breakdown in adipose tissue and glycogen breakdown in liver and muscle. Muscle, adipose tissue GluT-4 stimulated 10–20 fold. Liver GluT-2 insulin-independent, but Glc-metabolism stimulated.

569

31.1.2

Biochemistry and Genetics

Brain, RBC insulin-independent Glc-consumption. Glucagon is the most important functional antagonist of insulin in the liver. It has little effect on other tissues. Its plasma level is elevated during fasting and in hypoglycemia. It maintains an adequate blood glucose level during fasting. Epinephrine and norepinephrine are released during acute physical and psychological stress, including physical exercise, cold exposure, and biochemistry exams. They mobilize energy reserves (fat, glycogen) to cope with an increased demand. Although not involved in glucose homeostasis, they are released in hypoglycemia → Stress-symptoms. Glucocorticoids are elevated in chronic stress and act as gene-regulators. Adaption to life in a dangerous world: mobilization of reserves by stimulation of lipolysis, protein degradation, gluconeogenesis, glycogen synthesis. Glucagon, epinephrine, cortisol, and growth hormone are functional insulin antagonists. The excessive release of these hormones can cause hyperglycemia. Chronically ill patients: Muscle wasting, low Glctolerance, insulin resistance. Hormonal regulation of metabolism

Liver Glycolysis Gluconeogenesis Glycogen synthesis Glycogenolysis Fatty acid synthesis Transaminases β-Oxidation Muscle Glucose uptake Glycogen synthesis Glycogenolysis Glycolysis Protein synthesis Adipose tissue Glucose uptake Lipoprotein lipase Triglyceride synthesis Lipolysis

Insulin

Glucagon

Epinephrine

↑↑ ↓↓ ↑↑ ↓↓ ↑↑

↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑ ↑

(↓) (↑) ↓ ↑↑↑ (↓)

↑↑ ↑↑ ↓↓ ↑ ↑ ↑↑ ↑ ↑ ↓↓

Cortisol

↑↑ ↑

Allosteric activator

Allosteric inhibitor

AMP, ADP ATP, citrate Glucose 6-P

ATP, citrate AMP, ADP

Citrate

Glucose Acyl-CoA

↑↑ Malonyl-CoA Glucose 6-P AMP AMP ADP

↓↓ ↑↑↑ ↑↑↑

Glucose 6-P ATP H+

↓

↑↑↑

(↑)

2. Glucagon is the most important functional antagonist of insulin in the liver. It has little effect on other tissues. Its plasma level is elevated during fasting and in hypoglycemia. It maintains an adequate blood glucose level during 31.1.2. Metabolic role of organs fasting. 3. Epinephrine and norepinephrine are released during acute physical and psychological stress, keeps including physical cold exposure, and glucoLiver central organ of metabolism, blood glucoseexercise, constant: Glycogen storage, biochemistry exams. They mobilize energy reserves (fat, glycogen) to cope with neogenesis, nitrogen metabolism. All other organs “extra-hepatic” or “peripheral”. an increased demand. 4. Glucocorticoids are elevated in chronic stress. They mobilize reserves, including fat and protein, for energy and gluconeogenesis. Glucagon, epinephrine, cortisol, and growth hormone are functional insulin antagonists. The excessive release of these hormones can cause hyperglycemia. 570 III. THE STARVE-FEED CYCLE Adaptations to an intermittent food intake include: 1) The storage of excess metabolic energy it is plentiful, and its mobilization during fasting. 2) The maintenance of a stable blood glucose level, especially for the brain which has only a limited ability to use alternative fuels. The insulin/glucagon ratio is the most important hormonal factor for

31.3

The respiratory quotient

Kidney Considerable gluconeogenesis in kidney cortex during starvation (even short term), exertion (Cori-cycle) and acidosis. Substrates: lactate, glutamine and glycerol. Adipose tissue Storage of fat, fat synthesis from carbohydrate (limited). Muscle storage of glycogen and creatine phosphate only for itself (no Glc-6Pase!). Coriand Ala-cycle. Heart continuously active, aerobic metabolism only, Glc, fatty acids and ketone bodies used as fuel. Stores only limited amount of phosphocreatine. Brain uses O2 at constant rate, aerobic metabolism only, dependent on Glc, in starvation partly ketone bodies as their higher concentration in the blood allows uptake. No storage of glycogen. Fats can not cross blood/brain barrier. Blood transporter of nutrients, hormones and waste products. Erythrocytes only anaerobic Glc metabolism.

31.2. The respiratory quotient RQ is defined as the ratio of CO2 formed by O2 consumed. Burning of different compounds leads to different RQs: Glucose C6 H12 O6 + 6O2 * ) 6CO2 + 6H2 O

RQ = 6/6 = 1

Fat (Palmitic acid) C16 H32 O2 + 23O2 * ) 16CO2 + 16H2 O

RQ = 16/23 = 0.7

Protein RQ = 0.8 − 0.9 (empirical) RQ > 1 possible reasons • Growth • storage of fat • anaerobic metabolism At the same time different foodstuff contain different energy: Nutrient ∆H (kJ/g) CHO 17 Protein 17 Fat 39 Ethanol 30

571

31.3

Biochemistry and Genetics

Figure 31.1.: Ergospirometers are now very small and portable. Their main use is in sports and occupational medicine as well as in basic research (picture by the manufacturer).

572

The respiratory quotient

31.3

Figure 31.2.: Use of ergospirometry in sports medicine: The client is working out under a constantly increasing load (green). As a consequence, O2 consumption (blue) and CO2 production (magenta) increase up to a point where no more oxygen can be delivered to tissues, therefore oxygen consumption stays constant. The increased metabolic demand from the increasing load is met by anaerobic metabolism, resulting in a steeper increase of CO2 -production. Optimal training results are achieved if the workload is adjusted so that 60 % of the maximum oxygen consumption rate is used.

573

31.3.1

Biochemistry and Genetics

Table 31.1.: Energy stored in a well fed individual. Most of the energy is stored as fat, some as protein. Other energy sources have (quantitatively) only a minor role. Nutrient Organ Amount (kg) Energy (MJ) Triglyceride Adipose tissue 10–15 377–586 Protein Muscle 6–8 126–167 Glycogen Muscle 0.3 5.1 Liver 0.08 1.3 small molecules Blood 0.023 0.42

31.3. The Starve-Feed Cycle Adaptations to an intermittent food intake include: • The storage of excess metabolic energy if it’s plentiful, and its mobilization during fasting. • The maintenance of a stable blood glucose level, especially for the brain which has only a limited ability to use alternative fuels. The insulin/glucagon ratio is the most important hormonal factor for metabolic adjustments to the nutritional state. Typical blood levels: Analyte Very well-fed Insulin (U/mL) 40 Glucagon (pg/ml) 80 Insulin/glucagon 0.50 (ratio: U/pg) Glucose (mg/dL) 113 Fatty acids (mM) 0.14 Acetoacetate (mM) 0.04 β-Hydroxybutyrate 0.03 (mM)

Postabsorptive 15 100 0.15

3 days 8 150 0.05

89 0.6 0.05 0.10

70 1.2 0.4 1.4

Starved 6 weeks 6 120 0.05 67 1.4 1.3 6.0

Glucose homeostasis:

31.3.1. ofafter the meal): starve-feed cycle Phase I (0Phases – 4 hours Blood glucose is derived from dietary sources. Blood glucose is derived from dietary sources. Glucose uptake in muscle and adipose tissue and its • Phase I (absorptive, 0–4 h after meal): metabolism in most tissues are stimulated by insulin. Excess glucose is used for the synthesis of glycogen (liver, from muscle) and fatty acids (liver). All tissues use – blood glucose comes intestine. glucose as fuel. – glucose uptake in muscle, adipose tissue and liver (Km of glucokinase!)

Phase II (postabsorptive, 4 – 16 hours after meal): Glycogen breakdown in the liver is the major source of blood glucose. Hepatic gluconeogenesis starts. Phase III (16 – 48 hours after meal): 574 Liver glycogen is almost depleted. Blood glucose is from gluconeogenesis. Phase IV & V (>2 days after meal): Blood glucose is from gluconeogenesis only. During starvation, most tissues switch glucose to alternative energy sources: In phase II, the liver no longer consumes glucose. In phase IV and V, only brain, RBCs and renal medulla still use significant amounts of glucose. At

Phases of the starve-feed cycle

31.3.1

– glycogen synthesis in liver and muscle – lipid synthesis in liver (glucose) and adipose tissue (glycerophosphate from glucose, fatty acids from chylomicrons & VLDL) – Lipoprotein lipase in adipocytes but not muscle stimulated by insulin: chylomicrons routed to adipocytes – all tissues use glucose as fuel, liver, kidney and intestine also amino acids – Brain consumes 90–120 g/d of glucose – insulin/glucagon is high – luxus consumption: increased metabolism (postprandial thermogenesis) ≈10 %. Highest in unbalanced meals. • Phase II (post-absorptive, 4–16 h after meal): – blood glucose comes from liver glycogenolysis (about 50 %), and gluconeogenesis in liver (30 %) and kidney (20 %). – liver no longer consumes glucose – lipid synthesis in adipose tissue drops from lack of glycerol phosphate – glucagon ↑, insulin ↓ but still important • Phase III (16–48 h after meal): – adipose tissue lipoprotein lipase ↓, no uptake – hormone sensitive lipase becomes active, lipids from adipocytes are major fuel – liver glycogen almost depleted, blood glucose comes mostly from liver + kidney gluconeogenesis, which is fully active (amino acids, glycerol, lactate) – liver uses fatty acids from adipose tissue as energy source, glycerol for gluconeogenesis – liver releases ketone bodies into serum, about 150 g/d. β-oxidation of FA in liver generates energy for gluconeogensis – various organs (e.g. heart) use ketone bodies as fuel (i.e. perform the TCA-cycle) – respiratory quotient drops from 0.9 to 0.7 • Phase IV (starvation, 2–7 d after meal): – adipose tissue main source of metabolic energy, loosing 180 g/d of triglyceride – reduction of gluconeogenesis → saving of muscle mass

575

31.3.2

Biochemistry and Genetics

– blood glucose comes from gluconeogenesis only (150 g/d), muscle protein loss 75 g/d – only brain (↓ to 100 g/d), RBC (20 g/d) and renal medulla use glucose as fuel (uptake insulin-independent!). Consumption of ketone bodies by brain 50 g/d (30 % of energy) as their higher concentration in the blood allows uptake over the blood-brain barrier. – moderate reduction in basal metabolic rate (15–25 %, privo conservation). • Phase V (long-term starvation, several weeks after a meal): – muscle and many other tissues stop using ketone bodies in favor of fatty acids → blood [KB] ↑ – brain uses mostly ketone bodies (about 100 g/d ≡ 75 % of energy consumption). Glc consumption down to 30–40 g/d. – muscle protein loss reduced to 20 g/d – survival depends on fat reserves, not muscle protein (3–14 mo) – after depletion of fat, proteins are used as fuel → heart failure, death

31.3.2. Role of organs during starve-feed cycles During starvation, most tissues switch from glucose to alternative energy sources: In phase II, the liver no longer consumes glucose. In phase IV and V, only brain, RBCs and renal medulla still use significant amounts of glucose. At this point, the brain covers 50 % of its energy needs from ketone bodies rather than glucose. The reduction of glucose utilization spares protein, which would otherwise be degraded for gluconeogenesis. Glucose is redirected to brain, kidney medulla and RBCs because glucose metabolism in these tissues is independent of insulin.

Adipose tissue Adipose tissue synthesizes fat after meals, using glycerol phosphate derived from glucose and fatty acids from chylomicrons (after a fatty meal) or VLDL (after a carbohydrate meal). Fat synthesis is inhibited during fasting, mainly for lack of glycerol phosphate, and the hormone-sensitive lipase becomes active when the insulin level drops.

576

Role of organs during starve-feed cycles

31.3.2

Liver The liver oxidizes mostly amino acids after a mixed meal. 20–25 % of the dietary carbohydrate is metabolized in the liver. Most of this is used for the synthesis of glycogen and fat. Only a small fraction of the dietary fat is used by the liver because the liver doesn’t have LPL. During fasting, fatty acids from adipose tissue are the only important energy source for the liver. Ketogenesis is stimulated during fasting because of the ample supply of fatty acids and because the carnitine shuttle and the ketogenic enzymes are induced. The acetyl-CoA formed by β-oxidation is not used for biosynthesis because fatty acids synthesis and cholesterol synthesis are inhibited. Also TCA cycle activity is reduced because β-oxidation supplies enough NADH + H+ and FADH2 for the respiratory chain during fasting: the TCA cycle is not required to produce reduced coenzymes. Most people can survive for a few months without food, depending on fat stores. Refeeding of starved patients should be started slowly. There is severe glucose intolerance during extended fasting because the levels of glycolytic enzymes are very low. Especially in the case of kwashiorkor a protein-saving diet must be used, which contains enough carbohydrate and fat to meet the catabolic needs of the patient. Protein degradation after a protein-rich diet would overtax the capacity of an already damaged liver, possibly resulting in liver failure and death! The diet also needs enough protein to meet the anabolic needs of the patient, and this protein needs to be of high biological value. Otherwise the liver would have to catabolize unused amino acid and... see above! Re-feeding of starving patients is performed by the WHO 10-step scheme: hypothermia blankets (low subcutaneous fat!) hypoglycemia monitor blood [glucose], give oral or i.v. glucose dehydration if conscious: oral rehydration with higher K+ , lower Na+ (i.v. if not). Cave: heart failure! micronutrients give vitamins and trace elements infections give broad-band antibiotic (Cave: infections w/o fever!). Therapy against malaria if endemic. electrolytes K+ + Mg2+ starter nutrition SLOWLY! Protein-saving rather than protein-rich. tissue building rich diet dense in energy, protein and all essential nutrients that is easy to swallow and digest stimulation prevent as far as possible permanent damage by psychological, intellectual and motor activities

577

31.3.2

Biochemistry and Genetics

prevention of relapse identify cause of malnutrition, involve family and community in prevention. Cave: HIV! The basal metabolic rate decreases during extended fasting, but only to a modest extent. The respiratory quotient decreases during fasting as the tissues switch from glucose oxidation to fat oxidation.

Skeletal Muscle Fatty acids (50 %) and glucose are the major fuels for resting muscle after a meal, fatty acids and ketone bodies during fasting. Glycolysis, TCA cycle and oxidative phosphorylation operate at 10 % of capacity in resting muscle. Anaerobic exercise is heavy, short-term exercise like sprinting and weight lifting. Initially, ATP is generated in the reversible creatine kinase reaction: Creatine GGGGGGGGGGGGGB Creatine-phosphate + ADP F GG Creatine + ATP kinase Then, glycogenolysis and glycolysis are stimulated by low energy charge and cAMP (epinephrine). Blood flow to the muscles is poor, and there is little inter-organ cooperation. Anaerobic glycolysis is limited by acidity (lactic acid!) which inhibits phosphofructokinase. Aerobic exercise (swimming, long-distance running) relies on oxidative metabolism of: • Locally stored glycogen • Fatty acids from adipose tissue • Glucose and ketone bodies from the liver. Fat breakdown in adipose tissue and glycogen breakdown in muscle and liver are stimulated by epinephrine. There is insulin-independent glucose uptake in contracting muscle. The respiratory quotient may decrease during very mild physical activity, because mildly active muscles burn a lot of fatty acids. But it increases during vigorous exercise, because stored glycogen is oxidized. Lactic acid levels increase 5–10 fold during strenuous exercise. Carbohydrate loading can be used to increase muscle glycogen before athletic contests. This is important for endurance athletes, because muscle (and liver) glycogen lasts for 2 h during a marathon.

578

31.3.2

Role of organs during starve-feed cycles

Substrate cycles:

Substrate cycles .

Cori cycle (mostly during exercise):

Liver

Muscle

Glucose

Glucose 4 ATP 2 GTP

2 ATP

2 Lactate

2 Lactate

Alanine cycle (during fasting, also during exercise):

Liver

Muscle

Glucose

Glucose

4 ATP 2 GTP 2 NADH 2 Pyruvate Urea

2 ATP 2 NADH 2 Pyruvate α- Amino acid α- Ketoacid

2 Alanine

2 Alanine

In addition, there is the Gln cycle between muscle and kidney cortex. These cycles allow cycle allowsamino the muscles to oxidize acids of Gln. the the This muscles to oxidize acids and dispose of theamino nitrogen in theand formdispose of Ala and nitrogen in the form of alanine. During dasting, any glucose taken up in spite During fasting, any glucose taken up in spite of low insulin levels is recycled to the liver of as low alanine. insulin levels is recycled to the liver as alanine. Anabolic effects muscleprotein protein(more (more protein protein synthesized than degraded) onon muscle synthesized than degraded) Anabolic effects - Insulin - Growth hormone • Insulin - Androgens, anabolic steroids - Regular exercise • Growth hormone Catabolic effects: - Glucocorticoids - Protein-calorie malnutrition • Androgens, anabolic steroids - Denervation - space travel (weightlessness) • Regular exercise

The myocardium uses metabolic fuels similar to skeletal muscle, but there is more oxidative metabolism and less lactate formation. Lactate from skeletal muscle is oxidized in the heart during exercise. 579 V.

DETOXIFICATION REACTIONS AND ALCOHOL METABOLISM

252

31.3.2

Biochemistry and Genetics

Catabolic effects (degradation exceeds synthesis) • Glucocorticoids • Protein-calorie malnutrition • Denervation • space travel (weightlessness) The myocardium uses metabolic fuels similar to skeletal muscle, but there is more oxidative metabolism and less lactate formation. Lactate from skeletal muscle is oxidized in the heart during exercise.

Fuel use by the exercising muscle sprint stored ATP (2–4 s) and creatine phosphate (6–20 s) 800 m anaerobic glycolysis, using glycogen stores: Glc + 3 ADP + 3 Pi → 2 lactate + 3 ATP + 2 H2 O. Limited by acidification after ≈ 2 min. Cori-, Gln- and Ala-cycle. Fast-twitch, white muscle. 10 000 m aerobic oxidation of Glc (from muscle + liver glycogen stores), fatty acids (from adipose tissue) and ketone bodies (from liver) to CO2 . Rate limited by oxygen supply. Glc uptake no longer limited by insulin. Slow twitch, red muscle. 2–3 h. Marathon Runner “hits the wall” as muscle + liver glycogen stores are used up. Aerobic oxidation of fuels (FA, KB) delivered from liver and adipose tissue, amino acid catabolism. Insulin ↓↓, Glucagon ↑, catecholamines ↑↑ during exercise. Training increases blood supply, mitochondria (number + size), GluT-4 and fatty acid transporter, glycolytic enzymes, muscle mass per fibre.

Nutrition in athletes • plenty of protein after training to replace lost AA and build muscle mass • carbohydrate loading (70–80 % for 3–8 d) before competition to build up glycogen stores • no carbohydrates immediately before competition to lower insulin • sweet drinks during endurance exercise to prevent hypoglycemia

580

31.3.2

Role of organs during starve-feed cycles

Blood Cells Mature RBCs have no mitochondria. Glucose is metabolized by anaerobic glycolysis (ATP formation) and the pentose-phosphate pathway (NADPH + H+ ) formation. Only 20 g are consumed per day. Synthesis of 2,3-bisphosphoglycerate (BPG) . O

O C O

2PO3

HC OH C O H2

2-

PO3

1,3-bisphosphoglycerate

bisphosphoglycerate mutase

H2O

C OH

O

Pi

C OH

2-

HC OH

2-

C O H2

HC O PO3 C O H2

PO3

2,3-bisphosphoglycerate

2-

PO3

3-phosphoglycerate

2,3-bisphosphoglycerate is a potent inhibitor of bisphosphoglycerate mutase. Methemoglobin reductase is a flavoprotein that uses either NADH + H+ or NADPH + H+ to reduce methemoglobin back to hemoglobin: 2 Hb·Fe3+ + NAD(P)H → 2 Hb·Fe2+ + NAD(P)H + H+ Phagocytic cells rely on anaerobic glycolysis in the resting state but consume a lot of oxygen during work (“respiratory burst”). The intracellular killing of phagocytosed microorganisms can be achieved by: • Low pH of the phagocytic vacuole (= phagosome) • Lysosomal enzymes in the phagolysosome (created by fusion of phagosome and lysosome) • Oxidative reactions which create toxic superoxide radicals, hydrogen peroxide, and hypohalite: NADPH + Superoxide radical NADPH + H+ + 2 O2 GGGGGGGGGGGGGA NADP+ + 2 O− 2 + 2 H oxidase Superoxide +G Hydrogen peroxide 2 O− + 2 H G G G GGGGGGGGGGGGA O2 + H2 O2 2 Dismutase Myelo Hypohalite (hypochlorite, hypoiodite) Cl− + H2 O2 GGGGGGGGGGA OCl− + H2 O peroxidase An X-linked recessive deficiency of NADPH oxidase causes chronic granulomatous disease, with impaired intracellular killing and recurrent infections.

581

31.3.3

Biochemistry and Genetics

Brain Composition Water 70 % of white matter, 80 % of gray matter Lipids 60 % of dry weight in white, 40 % in gray matter Myelin contains 80 % of dry weight lipid. Galactocerebroside and -sulfatide are mostly in myelin, gangliosides mostly in the gray matter. Metabolism Glucose oxidation is the principal energy source except in prolonged fasting when ketone bodies are also important. The brain accounts for 25 % of the basal metabolic rate in adults and > 50 % in infants. Insulin has no effect on brain carbohydrate metabolism, except in the ventromedial hypothalamus (satiety center!). Brain activities such as perceiving, feeling, remembering, and reasoning cause moderatesized increases of brain metabolism in circumscribed brain regions. These metabolic changes can be picked up in functional imaging studies (PET-scan). Metabolic insults Oxygen deprivation causes loss of function within seconds, followed by rapid cell death. In adults, irreversible brain damage occurs after about 7 min in drowning victims. Newborns develop irreversible brain damage after 1 h of oxygen deprivation, with cortical cell loss. Hypoglycemia causes weakness, trembling, facial flushing, sweating, in severe cases progressing to confusion, seizures, and coma. The brain stores only very small amounts of glycogen. Hyperglycemia is probably not damaging itself, but is usually accompanied by acidosis and dehydration which cause CNS depression in patients with “diabetic coma”. Uremia, acidosis, hyperosmolarity, and hyperammonemia all result in encephalopathy. The blood-brain barrier has carriers for the facilitated diffusion of glucose, amino acids etc... Cerebrospinal fluid, formed in the choroid plexuses, has: • an electrolyte composition similar to plasma. • a very low protein concentration (20 mg/dL). • a glucose concentration that is 65 % of blood glucose. Lumbar CSF is important for the diagnosis of CNS diseases.

582

Obesity

31.4

31.3.3. Unbalanced meals A high carbohydrate/low fat meal stimulates lipogenesis in the liver. VLDL and plasma triglycerides increase well above the post-absorptive level. In short-term experiments, sucrose elevates plasma triglycerides more than complex carbohydrates, probably because fructose is more rapidly degraded than glucose (no glucokinase and phosphofructokinase required). A high protein/low carbohydrate meal stimulates insulin release, but glucagon release is also stimulated and this prevents hypoglycemia. A high-fat/low carbohydrate/low protein meal leaves insulin low and glucagon high, with very high plasma levels of free fatty acids and ketone bodies. This kind of diet (Atkins diet) has been recommended for weight reduction and may indeed be “effective”: to maintain the blood glucose level, a lot of muscle protein has to be degraded to supply amino acids for gluconeogenesis. Ketone-bodies produced from ketogenic amino acids have a diuretic effect, leading to loss of water. Postprandial thermogenesis is highest for protein, lowest for fat and intermediate for carbohydrates. It is higher after a very unbalanced (carbohydrate only/no fat or fat only/no carbohydrate) than after a well-balanced meal.

31.4. Obesity Obesity is a chronic disease caused by imbalance between energy supplied with meals and required by resting metabolism + physical activity. Body weight usually builds up over long-terms: • average | requires 12 000 kJ/d. • said | exceeds requirements by 1% on average, 120 kJ/d (Lipostat theory) • 1 kg of fat is equivalent to 32.3 MJ • thus the | gains 1.36 kg/a in body fat • over 20 years that adds up to 27 kg! There are large differences in individual energy requirement, which make it difficult to determine an appropriate caloric intake: • physical activity • resting metabolism (30 % individual difference after adjustment for age, sex, size)

583

31.4.2

Biochemistry and Genetics

Rapid weight gain in is observed in some serious diseases (hypothyroidism, Cushing disease) and always needs to be investigated! Since obesity is caused by an imbalance between ingested and spend energy, it is, in principle, curable by reducing energy input and/or by increasing energy expenditure. In practice however, this is much easier said than done. Several causes conspire: • Until less than 50 a ago most people had to work hard physically to make a living. Our digestive system is adapted to deliver 2–3 times more energy than required for a modern sedentary lifestyle. • During much of human evolution food supply was highly irregular. We evolved a tendency to store energy for meagre times, details of which will be discussed in the following sections. With a fast food store at every corner this ability has become a liability. • With fewer and fewer people having the ability, interest and time to prepare their own meals dependency on industrially prepared food with a lot of protein, fat and salt, but little fibre has increased. • Portion size of prepared food has increased, “supersize” • There is an effect of social contagion: The more people in a patients environment are obese, the more likely the patient is to develop obesity. It just “seems ok”.

31.4.1. Adipose tissue in obesity • Normal person 2 × 1010 fat cells each storing 0.3 µg of fat (6 kg total) • Max. capacity 0.9 µg per fat cell, 18 kg total • If this amount is exceeded, fat cells divide to accommodate extra fat → obesity, “adipose tissue hyperplasia” • Obese person, say, 8 × 1010 fat cells with 0.9 µg → 72 kg body fat • after weight reduction still 8 × 1010 fat cells, but with, say, 0.2 µg of fat (16 kg body fat) • Underfilled fat cells signal: “Feed me!” → jojo-effect • Reduction in BMR like in starvation Thus it is the adipose tissue hyperplasia that defines obesity, BMI and waist/hip-ratio are only tools to quickly assess a patient.

584

Adipose tissue in obesity

31.4.2

Figure 31.3.: In a healthy environment both people resistant and susceptible to a multifactorial disease do quite fine. Once the environment turns toxic, susceptible people fare far worse then resistant ones.

585

31.4.4

Biochemistry and Genetics

31.4.2. Appetite control • Stomach stretch receptor • Blood glucose sensor • Intestinal hormones • Adipose tissue: Leptin (from Greek: thin, 167 aa protein) – LepRb in ventromedial nucleus of the hypothalamus: satiety center, endo-cannabinoids ↓, vegetative NS ↑ – high serum [Leptin] prevents active transport of leptin across blood-brain barrier: leptin insufficiency – desensitization of LepRb by high [Leptin] in obesity: Leptin resistance, e.g. in heart muscle – inherited deficiency in leptin or its receptor in some obese people (see fig. 31.5) – fertility regulation by leptin: amenorrhea in anorexic ~ – many other functions

31.4.3. Role of gut flora • 1013 to 1014 symbiotic bacteria on humans, most of them in the end-gut. • Digest fiber into acetate, propionate, butyrate → 10 % of total energy intake and principle food for colonocytes. • Synthesis of essential aa, vitamins (but recall that their uptake is mostly in the small intestine!). • detoxification of plant secondary metabolites (cancer protection?) • Out of 70 eubacteria and 13 archaea divisions only 2/1: Bacteroidetes, Firmicutes and Methanobrevibacter smithii make 93 % of gut biodiversity. Note: E. coli occurs in the highest number, but is a γ-proteobacterium, a group that does not contribute much to diversity. • In obese people and ob/ob mice B/F ratio drops, but biodiversity stays constant. • B/F ratio can be regenerated by weight reduction. • Does bacterial fauna influence how well we use food?

586

31.4.4

Role of gut flora

Figure 31.4.: Model for the lipostat: If the amount of fat in adipose tissue increases, this releases leptin into the blood stream. Leptin stimulates the release of various hormones in the hypothalamus, which all reduce the production of neuropeptide Y. This reduces the inhibitory effect of NPY on the production of thyroid hormone, the increased thyroid hormones lead to increased energy expenditure and hence decrease fat stored in the adipose tissue. In addition, NPY reduces feeding behavior, this too reduces fat stores. The system is set for a slow weight gain, which made sense in a world with irregular food supply. Leptin

Lipostat theory Hypothalamus

propiomelanocortin (POMC)

corticotropin releasing hormone (CRH)

cocaine and amphetamine regulated transcript (CART)

melanocyte stimulating hormone ( MSH)

neuropeptide Y (NPY)

Thyroid hormone

energy expenditure

feeding behaviour

Thermogenesis Adipose tissue mass

587

31.4.4

Biochemistry and Genetics

Figure 31.5.: Left: Inherited leptin deficiency in a 3 a old boy leads to morbid obesity. Such patients show aggressive feeding behavior (stealing from table neighbors or garbage cans, raiding fridges). Right: The same boy after 4 a treatment with recombinant leptin. Figure from [O´Rahilly et al., 2003].

588

Epigenetics

31.4.5

31.4.4. Beneficial effects of dietary restriction • life expectancy highest in people about 10 % underweight • Class-III protein deacetylases (sirtuins) are NAD-dependent and measure NAD/NADH ratio. • act on: histones, p53,... • In yeast silent mating type information regulator 2 (Sir-2) – human ortholog SIRT-1 – activated by polyphenols: beneficiary effect of red wine in low doses

31.4.5. Epigenetics Until a few years ago evolution was assumed to be a slow process of accumulating mutations in the DNA by a trial and error process. Recently it has turned out that the expression of genes can be regulated by methylation of their DNA on the cytosine at CpG dinucleotides or chromatin remodeling (mostly by histone acetylation, but also methylation, phosphorylation, ubiquitinylation, SUMOylation, ADP-ribosylation, biotinylation), and that these changes are inherited. This allows rapid adaptation to a changing environment – and the human environment has changed a lot over the last 100 a. In a way, this looks a bit like theories of inheritance proposed by Lamarck. Information that is inherited other than in nucleic acid sequences is called epigenetic, this includes for example the structural information of a living cell – which is why cells can originate only from cells, not from just DNA. Unfortunately our understanding of epigenetic phenomena is still rudimentary. Some observations may indicate how important this field will become: • In a study on harvest records and death certificates in Överkalix, a remote village in Sweden, researches found that if grandfathers had been exposed to famine during adolescence, their grandchildren had longer life expectancy and a lower incidence of type II diabetes. If the grandmothers had been exposed to famine during embryonal development the grandchildren had a higher incidence of low birth weight and of type II diabetes. Think: Why is the sensitive period in males prepuberty and in females embryonal development? This field is called transgenerational epigenetic inheritance. • Exposure to famine in utero seems to set a thrifty genotype by epigenetic mechanism that is adapted to malnutrition. If later in life the nutrition is much richer, the body is unable to handle it, resulting in metabolic disease. Similarly, if nutrition in utero is rich, the person will find it more difficult to survive famine later in life. Low birth weight has been shown to be a risk factor for glucose intolerance.

589

31.4.5

Biochemistry and Genetics

Figure 31.6.: Deacetylases come in 3 different classes. While class I and II use water, class III deacetylases use NAD+ . Because the NAD+ /NADH + H+ ratio depends on the nutritional state, class III deacetylases can regulate cellular processes (like division) depending on food supply. Figure from [Buxbaum, 2007]. H

+ NH3

+H2O

N

CH3


  

      

+

O

CH3 O


  

O

acetate

N-acetyl-lysyl-group

N

N N

N

O

O

O P

O

O
  

   

NH2

O

NH2

HO


  

O

O

P

O

+ N

O HO

OH

OH

NAD+

NH2

+

NH3

N

NH2

O

+

+

N N

N

O

O

N

O P O

HO

OH

O

O P

O

OH

O HO

Nicotinamide O-acetyl-ADP-ribose (second messenger)

590

O

O

O CH3

Systems biology

31.4.6

Figure 31.7.: Each gene gives rise to several mRNAs by alternative splicing, RNA editing and the like. The collection of mRNAs present is called the transcriptome of a cell. Each of these mRNAs gives rise to different protein species by posttranslational modification, the collection of proteins is called the proteome. Interactions between proteins results in the interactome of a cell. By enzymatic activity these proteins change the composition of the cell with respect to small molecules, the metabolome. This in turn has effects on the physiological (and pathological) state of the cell, the phenome. Each of these levels of organization can be investigated with specialized methods. Figure from [Buxbaum, in press]

• Although the incidence of obesity in industrial nations is still increasing, the incidence of coronary heart disease and type II diabetes is starting to decrease (“obese but metabolically healthy”). In developing countries, where the obesity epidemic is just starting, incidence of obesity and both diseases increase together. • Rats neglected by their mothers during infancy become timid and nervous compared to those raised by caring mothers. Drugs that facilitate the removal of methyl-groups from DNA in the brain can normalize their behavior. • Imprinting allows paternal and maternal genes to be distinguished and differentially expressed (or silenced) during development. During germ cell development these imprinting patterns are erased and reestablished. • Since epigenetic regulation is influenced by environmental factors like nutrients (folic acid and methionine, individual and maternal diet during pregnancy) the variable penetrance of some genetic diseases can be explained. This offers a theoretical route for the treatment of affected patients. For a review on these topics see [Mariman, 2008].

591

31.4.6

Biochemistry and Genetics

Figure 31.8.: If genes are sorted by interaction of their products large clusters are revealed. It is the proteins of these those clusters that work together for a particular effect. Figure taken from [Sieberts and Schacht, 2007].

592

Systems biology

31.4.6

Figure 31.9.: A particular disease state like atherosclerosis involves several biochemical pathways, each consisting of several proteins. Figure taken from [Ghazalpour et al., 2004].

593

31.5

Biochemistry and Genetics

31.4.6. Systems biology Originally biochemistry dealt with the working of a particular protein, say, an enzyme. Later these enzymes were grouped into pathways, and we learned that in a pathway more features could be observed that in a single enzyme (e.g. end product inhibition). The whole is more than the sum of its parts. Today we try to understand the entire complexity of the interaction of various pathways. This is the aim of systems biology. Long since the complexity of the information exceeds human understanding, only with the help of evermore powerful computers can we hope to make head and tail of it. Only 100 a ago visitation by god was an officially recognized diagnosis on death certificates. In 20 years a diagnosis like metabolic syndrome will probably look equally quaint, having been recognized as a hodgepodge of different diseases with quite different treatment.

31.4.7. Metabolic syndrome Definition (International Diabetes Foundation 2005) central obesity waist circumference > ethnic + sex specific limit plus any two of raised triglycerides > 150 mg/dl (1.7 mM) reduced HDL cholesterol < 40 mg/dl (1.03 mM) in ♂, 50 mg/dl (1.29 mM) in ♀

raised blood pressure ≥ 130/85 mmHg

hyperglycemia fasting blood [glucose] ≥ 100 mg/dl (5.6 mM): Oral glucose tolerance test recommended, but not required for diagnosis of metab. syndrome. previously diagnosed diabetes Waist circumference limits (cm): Ethnic group ♂ ♀ Ethnic group use Europids 94 80 South + Central Americans South Asian South Asians 90 80 Sub-Saharan Africa European Chinese 90 80 Eastern Mediterranean European Japanese 85 90 Arab European Insufficient data on some populations, use limits of comparable (!?) population for the time being.

594

31.5

Diabetes

31.5. Diabetes Type I insulin deficiency after destruction of pancreas β-cells by auto-immunity. Juvenile onset. Insulin-dependent. Prevalence 1:400, not life-style dependent. Patients are thin (wasting of fat and protein stores). Ketonemia and -uria, autoantibodies against islets. Type II insulin resistance in obesity. Adult onset. Prevalence 7:100 in the USA, costing 1 × 1011 US$ (≈ 6 % of total health care spending). Associated with hypertension, dyslipidemia, atherosclerosis (metabolic syndrome) • lower number of insulin receptors • lower affinity of receptors for insulin • interference with signalling cascade Therapy by weight reduction restores insulin responsiveness. Problem: Compliance. MODY see later.

Patho-mechanism of Type-2 Diabetes • insulin has anti-inflammatory effects, insulin resistance is pro-inflammatory • food intake → steeper H+ -gradient across inner mito membrane → ROS production in Complex II • NADH/NAD+ ↑ + NADPH/NADP+ ↓ → glaucoma

→ polyol and hexosamine pathways ↑

• Adipose tissue generates hormones + pro-inflammatory cytokines (obesititis): – leptin, adiponectin, visfatin – tumor necrosis factors α – transforming growth factor β, Insulin-like growth factor + binding protein – Plasminogen activator inhibitor 1, angiotensinogen – complement C3, IL-1, IL-6, IFN-γ – serum amyloid Consequences (“chronic acute phase response”): Adipocytes insulin resistance, free fatty acid release Liver release of acute phase proteins and lipids

595

31.5

Biochemistry and Genetics

Figure 31.10.: Uterus and ovaries of a normal (left) and a type I diabetic (right) rat. Note the absence of adipose tissue and the reduction of the organs. Figure from the archives of E.B.

596

Diabetes

31.5

Brain somnolescence, stimulation of Hypothalamus-Pituitary-Adrenal axis: corticosteroids and catecholamines, leucocytosis, sympathetic activation vascular endothelia activation by cytokines, steroids and catecholamines → hypertension. Microvascular damage → complications. muscle insulin resistance by Ser-phosphorylation of IRS-1 and -2 (via NFκB, IKK, JNK1?) gonads lowered sex hormone production, impotency in ♂ pancreas reduced β-cell function

Note that in “obese but metabolically healthy” persons the production of pro-inflammatory hormones is not increased! Maturity onset diabetes of the young (MODY, #606391, monogenic diabetes) • insulin resistant diabetes with early onset, usually < 25 a • no obesity or metabolic syndrome • high frequencies in Romania and African Americans • autosomal mutations of – hepatocyte nuclear factor HNF-4α (MODY-I, #125850), chromosome 20 and HNF-1α (MODY-III, #600496). Mild diabetes, progressive decrease of insulin secretion. – glucokinase (MODY-II, #125851), 130 different mutations, chromosome 7, mild glucose intolerance in heterozygotes with risk of gestational diabetes – insulin promotor factor (IPF-1). Pancreatic agenesis in homozygotes, MODY-IV (#606392) in heterozygotes. – β-cell transcription factor neuroD1 (MODY-VI, #606394) – carboxyl-ester lipase gene (MODY-VIII, #609812): diabetes with dysfunction of exocrine pancreas – phosphoenolpyruvate carboxykinase (PEPCK)1 (+261680): no down-regulation by insulin – mito DNA (OxPhos ↓, mitochondrial diabetes) – insulin receptor, GluT4, hexokinase II, SUR-1, insulin, IRS-1, PC-1, glycogen synthase

597

31.5

Biochemistry and Genetics

20

15

10

5

0

[blood glucose] (mg/dl)

(mM)

Figure 31.11.: The glucose tolerance test. After ingestion of 75 g of glucose on an empty stomach the blood glucose concentration is measured repeatedly over 2 h. In normal persons blood glucose concentration raises slightly and returns to normal quickly. In diabetics, the raise is much higher and the return to normal slower. diabetic 300

200

normal range

100

0 0

30 60 90 120 time after glucose feeding (min)

redrawn from Meisenberg & Simmons, 2006

Diagnostic • random blood glucose ≥ 11.1 mM (200 mg/dl) plus symptoms • fasting blood glucose level > 7.0 mM (126 mg/dl) on two occasions • Kidney threshold for glucose 10 mM (180 mg/dl), no threshold for ketone bodies • If 6.1 mM (110 mg/dl) ≤ fasting [glucose] ≤ 7.0 mM (126 mg/dl): Oral glucose (75 g) tolerance test: →≥ 11.1 mM (200 mg/dl) after 2 h, impaired glucose tolerance if [glucose] ≥ 7.8 mM (140 mg/dl) • Glycated hemoglobin (HbA1c ) 2.8–3.7 % in normal, > 6 % in diabetic patients. Integration of [glucose] over life time of erythrocytes (≈ 120 d). Note: Value is methoddependent, Labs need to report reference ranges with the result. The ranges here are for measurement according to current standard of the Int. Fed. of Clin. Chem. & Lab. Med. • Further info: Clin. Chem. Lab. Med. 41 (2003) Reviews on Diabetes (available in the library).

598

take blood sample

Yes

blood sample taken after > 8 h fasting?

no

Diabetes

no diabetes

fasting blood glucose > 7 mM

no

Symptoms?

Yes

no

determine fasting BGL

Yes

Yes

fasting blood glucose < 6.1 mM

> 11.1 mM

random blood glucose

no

Yes

oral glucose tolerance test

Diabetes

no diabetes

< 7.8 mM

2 h blood glucose

> 11.1 mM

Diabetes

7.8..11.1 mM

Reduced glucose tolerance

Diabetes

31.5

Figure 31.12.: Flow-diagram on the diagnosis of diabetes and reduced glucose tolerance.

599

31.5

Biochemistry and Genetics

Figure 31.13.: Glucation (non-enzymatic! ) on N-terminal Val of Hb-β. Initially an unstable Schiff-base is produced, which slowly turns into the relatively stable ketosamine. Upon heating this turns into caramel, which gives roast food its aroma. In the body, the ketosamine may be further processed into AGEs via Strecker degradation. HC

O

OH

COOH

+

HC OH

COOH H

+

H2O

+

HC N CH H HC OH R

H2N CH R'

R

HC N CH HC OH R'

R

R Immonium

Schiff base

COOH

CH2OH

COOH

O

H N CH

OH

H

R'

OH

+

HC N CH H C OH R' R Amadori-Rearrangement

OH

Glucosylamine

H2O C H2C N C H C O R' R Reductone (Caramel)

600

H COOH

COOH

O

H2C N CH H C O R' ! Maillard-Reaction

R

ketosamine

Strecker degradation

Diabetes

31.5

Glycation, AGE, ALE There are several modified hemoglobins in our blood: HbA1a glucose phosphate + fructose phosphate HbA1b pyruvate HbA1c glucose, ≈ 80 % of HbA1 Of those, only HbA1c is of diagnostic importance. From its concentration the average blood glucose level over the last 3 months can be calculated: [glukose](mM) = 1.84 × HbA1c (%) − 0.01 Again, this equation works only if the HbA1c was determined according to Int. Fed. of Clin. Chem. & Lab. Med. guidelines. The following advanced glycation + lipoxydation end-products (AGE/ALE) are important: Amadori-reaction aldose + amine → ketosamine (see fig. 31.13). ALE by similar reactions with lipid peroxides Lys + Arg adducts CML, CEL, GALA, LOMA, Pyrraline, CMA, Argpyrimidine, ... Lys-Lys crosslinks GOLD, MOLD, Crossline, Fluorolink, Vesperlysine Lys-Arg crosslinks Glucosepane, Pentosidine, GODIC, MODIC Modification of nucleophilic aa His, Trp, Ser, Cys Analytical procedures (ELISA) have been developed for total AGE/ALE and for CML and pentosidine. However, the interpretation of these values is currently unclear, the assays are used only in research. For that reason you may forget about all the acronyms above. • Concentration of AGE/ALE too low to directly affect enzymatic function • AGE-receptor (RAGE) on macrophages → inflammatory mediators →ROS-formation → damage • Correlation between collagen-AGEs and secondary diabetic damage, uremia, liver cirrhosis, arthritis, coronary artery disease, smoking. • Autofluorescence of connective tissue.

601

31.5

Biochemistry and Genetics

Catalogue of investigations for diabetics • First visit: – Complete physical exam (chronic complications?) – Neurological and angiological exam – Exam of feet – EKG – Eye exam (referral to ophthalmologist) – Blood sugar, HbA1c, Cholesterol (total, HDL, LDL, TG), creatinine, electrolytes – Urine sample (Glucose, Albumin (microalbumiuria 30–300 mg/d), Ketone etc.) – Start of: ∗ Agreement on short- and long term aims ∗ Structured training of patient / family ∗ Training in self exam (see below) ∗ Individual nutrition advise (nutritionist) ∗ Advise on healthy life style ∗ Pharmacological management ∗ Treatment of acute problems ∗ Contraceptives / pregnancy ∗ Next appointment • Each visit (follow up): – weight – blood pressure – blood glucose – Present condition and therapy (results, measures, aims) – Continue training • Every 3 months: – weight – blood pressure

602

Diabetes

31.5

– blood sugar – HbA1c – Lipids (only when elevated) – Urine for albumin (only when pathologic) – training with diabetes team • Annually: – complete physical (see first visit) – complete lab (see first visit) – check self-exam techniques Patient self exam for diabetics (daily): • Blood glucose • Acetone (if BG >250 mg/ dl / 13,8 mM) • Urine glucose • Weight • Blood pressure • Foot exam • Documentation in a log book! Complications of diabetes and their management • Hyperglycemia + ketoacidosis (rapid breathing, breath smells of acetone). Treatment: – Fluid replacement – correction of acidosis – insulin injection • Hypoglycemic shock in insulin-injecting patients if they fail to eat regularly. • long term damage: microvascular damage neuropathy

603

31.6

Biochemistry and Genetics

impaired wound healing monitor feet to prevent amputation retinopathy monitor retina, laser surgery where needed glaucoma monitor eye pressure, medications, (laser-)surgery nephropathy monitor microalbuminuria, use angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor blocker (ARB) to treat cardiovascular disease tight blood pressure control

31.6. Example questions Wasting in terminal illness X.Y. is in the end-stage of cancer. Upon a routine exam you find that he shows muscle and adipose tissue wasting. Following up on this finding you diagnose a reduced glucose tolerance combined with insulin resistance. Increased concentrations of which of the following hormones is responsible for the complication? A) Glucagon B) Insulin C) Epinephrin D) Thyroid hormone E) Cortisol

Obesity Which of the following is not usually associated with obesity? A High levels of leptin B Low levels of leptin C Changes in gut microbial fauna D Amenorrhea in females E Reduced sirtuin activity

604

Objectives in Summary

31.7

Metabolic syndrome A male patient comes to you with a waist circumference of 150 cm. Which of the following signs is he least likely to show? A) raised triglycerides (≥ 150 mg/dl or 1.7 mM) B) raised HDL cholesterol (≥ 100 mg/dl or 2.6 mM) C) raised blood pressure (≥ 130/80 mm Hg) D) raised fasting blood glucose (≥ 100 mg/dl or 5.6 mM) E) previously diagnosed diabetes

31.7. Objectives in Summary At the end of this lecture students should be able to • describe the role of insulin, glucagon, epinephrin and glucocorticoids in the control of metabolism. • discuss the use of macronutrients and their flow between various organs in the well-fed and fasting state. • describe how nutrients are used during physical exercise. • define RQ and describe how it can be used clinically • discuss the epidemiology, patho-mechanism, health consequences and treatment options of obesity. Describe obesity as chronic disease and state the lipostat theory of weight maintenance. • critically evaluate modern theories on mechanism and treatment of obesity. • define “metabolic syndrome” • describe the causes, clinical manifestations (especially in emergency room setting) and treatment options of diabetes type 1 and 2. • name secondary diabetic complications, their detection and treatment. • critically discuss the role of AGE/ALE in the development of diabetic complications.

605

32. Multifactorial Inheritance 32.1. Definitions Multifactorial traits are caused by “many factors”, both genetic and environmental. Polygenic means almost the same, implying the action of “many genes” but no environmental influence. Most of the contributing genes are still unknown. A trait is assumed to be polygenic or multifactorial if: • It is more common in close relatives of the index case than in the general population even if they were raised in uncorrelated environments, but • A Mendelian pattern of inheritance cannot be identified and • There is no recognizable chromosome aberration. Multifactorial traits include: • Most differences between normal people, including physical traits (height, weight, blood pressure) and psychometric traits (intelligence, personality). These traits show continuous variation. • Many common diseases, including diabetes mellitus, allergies, autoimmune diseases, cardiovascular diseases, neuropsychiatric disorders, and even infections. • Most congenital malformations. Our models for explaining multifactorial traits assume that the gene effects take on one of the following forms: Additive gene effects are effects of individual alleles. Additive inheritance assumes codominance. Also the phenotypic effects of alleles in different loci are assumed to be additive. This mode of inheritance predicts that close relatives should have similar phenotypes, in proportion to the genes they share. Non-additive gene effects are the effects of gene combinations, rather than individual alleles. Nonadditive effects within a locus include dominance, recessivity, and heterozygote advantage. Nonadditive interactions between genotypes in different loci are called epistasis and would for example include effects of mutations in a transcription factor that led to changes in expression of one allele in the target gene, but no

607

32.2

Biochemistry and Genetics

changes in expression of another allele in the same target locus. Non-additive inheritance does not necessarily predict similarity between close relatives, except identical twins who share not only individual alleles but all gene combinations.

32.2. Quantitative Traits If many loci determine the phenotype, the effects are additive, and each locus contributes only a small proportion of the total variability, we get a smooth bell-shaped distribution of phenotypes. This is called a normal distribution or a Gaussian distribution.

The standard deviation σ is a measure of the variability of the observed phenotype. In a distribution, 50 % of individuals are within ± 0.68 σ of the mean (x), 95 % are within 1.95 σ. The variance (V ) is the square of the standard deviation (V = σ 2 ). A bell-shaped distribution can be produced by many loci with 2 co-dominant alleles each, or by multiple alleles in one or a few loci. Broad heritability (H 2 ) is the proportion of the total phenotypic variance that is due to heritable factors. It can range from zero (only genes are important). Narrow-sense heritability (h2 ) is the proportion of the total variability that is caused by additive gene effects. The environmental variance can be partitioned into shared environment (factors that make members of the same family similar) and non-shared environment (factors that make members of the same family different). Examples of shared environmental effects are the education and economic conditions of the parents; most non-shared environmental effects originate outside the home. Most or all of

608

Quantitative Traits

32.2

the environmental variance is non-shared in adults. The various genetic and environmental effects are assumed to add up: VPhenotypic = VGadd + VGnonadd + VEshared + VEnonshared

(32.1)

Measurement error appears as part of the non-shared environmental variance. Therefore accurate measuring instruments have to be available, otherwise you over-estimate the importance of non-shared environment and under-estimate everything else. Assortative mating artificially increases the shared environmental variance. Heritability differs in different populations because the genetic and environmental variability are not the same in different populations. For quantitative traits, the average phenotype of a child is between the “midparent” and the population mean. This tendency for children to have less extreme phenotypes than their parents is called regression to the population mean. This effect is really only notable for the most extreme parents, and the explanation is that the parents have their extreme phenotype due to environmental rather than genetic factors. The greater the additive genetic + shared environmental variance, the lower is the regression to the mean. In adoption studies, regression from the phenotype of the biological parents to the population mean can be used to estimate the additive genetic variance. Note that the “population mean” differs in different populations. The children of unusually tall pygmy parents, for example, regress to the mean for pygmies, but children of equally sized (unusually small) non-pygmy parents regress to their own population mean. Height (stature) shows a normal distribution with a slight hump at the lower end, which is due to pathological conditions reducing height. Heritability is high (≈ 90 % in most studies). In Western countries, height increased by 5–10 cm during the past century, due to environmental changes (nutrition?). Differences between populations in different parts of the world are in part genetic and in part environmental. Body weight has a high heritability, but less than stature: ≈ 70 % in most studies. As in the case of height, the high heritabilities are obtained with reasonably homogeneous populations where environmental inequalities are not exceedingly large. Skin color differences between races are determined by about 3–5 major genes. Most of the gene products are unknown. Skin color differences evolved for protection from UV damage and the regulation of vitamin D synthesis. Test intelligence has a bell-shaped distribution with a hump at the lower end. In IQ tests, the population mean is defined as 100, and 15 points are one standard deviation. Broad heritability is typically about 40 % in children and 70 % in adults. The average IQ has increased by about 30 points during the past century because of environmental changes (better nutrition or education).

609

32.3

Biochemistry and Genetics

Mental retardation has heterogeneous causes: • Most cases of “mild” mental retardation (IQ 50–70) are multifactorial. This means that many relatives of mildly retarded individuals are mentally subnormal. • Mental retardation with IQ < 50 usually has a specific cause, such as an infection, perinatal hypoxia, fetal alcohol syndrome, kernicterus, trauma, severe epilepsy, a chromosome aberration, or a Mendelian disorder (most important: fragile X). In about half of retardates, however, no specific cause can be identified. Most etiologies are one-time accidents, therefore close relatives are usually normal. • Other multifactorial traits with continuous variation include personality traits with moderately high heritabilities (happiness, divorce liability...) or rather low heritability (love styles, self esteem...). Also athletic performance, age at first menstruation, age at first sexual intercourse, life span etc. are multifactorial traits.

32.3. Common Diseases Most diseases encountered in medical practice are “heritable” in the sense that they are more common in close relatives of a patient than in the general population. Typical features of multifactorial diseases: • The concordance between monozygotic twins is higher than between dizygotic twins of the same sex. But even concordance between monozygotic twins is rarely 100 %. • The disease incidence in first-degree relatives of a patient is close to the square root of the population incidence. • The risk for close relatives increases with increased severity of the disease in the index patient, and with the presence of more than one affected family member. These features suggest an especially bad accumulation of predisposing genes in the family. Note, however, that many diagnostic labels (“schizophrenia”, “diabetes”) are not a single disease. In these cases, the more severe forms do not always have a higher recurrence risk. • The recurrence risk decreases rapidly if the relationship is less close. The cutoff between “normal” and “affected” is often arbitrary. Therefore, a “disease” may simply be the extreme end of a normal distribution. Genetic counseling for multifactorial diseases is based on the empiric risk that is known from observations of a large number of families in the same situation. Diabetes Mellitus is a typical example:

610

Congenital Malformations (“Birth Defects”)

32.4.1

Type I diabetes is probably an autoimmune disease that can, most likely, be triggered by viral infection. Concordance in monozygotic twins is 30–50 %. It is associated with certain HLA-haplotypes (haplotype = combination of linked genes). The HLA antigens regulate immune responses. Type II diabetes has no HLA-associations, but the heritability is higher than for type I, with almost 100 % concordance in monozygotic twins. The risk in first-degree relatives of those patients who develop type II diabetes late in life (> 60 years) is not much increased above the population mean, but relatives of those who develop the disease early (< 40 years) are not high risk. One of the important risk factors for this type of diabetes is obesity. Genes predisposing for this type of diabetes seem to have been selected for in populations living in regions with a high risk for frequent starvation. When people from these populations move to areas where they live at “westernized” standards they have a high incidence of diabetes type II. Psychiatric disorders are usually multifactorial. In schizophrenia, with a population incidence of 1 %, the empiric risk for first-degree relatives is about 10 %. The empiric risk in children of two schizophrenic parents is about 30 %, and concordance between monozygotic twins is about 50 %. In cases with a negative family history, advanced paternal age is a risk factor. This suggests that new mutations are important. Also, for manic-depressive disease, primary epilepsy, Alzheimer’s disease, autism, attention deficit disorder, alcoholism, and personality disorders are multifactorial. Infections have substantial heritability in those cases where most or all members of the population are exposed to the disease agent. This has been shown for malaria, hookworm, tuberculosis, and other infections. The immune system is highly polymorphic by design, and therefore people vary greatly in their susceptibility to different infections.

32.4. Congenital Malformations (“Birth Defects”) About 1 in 40 babies are born with a congenital malformation (congenital = present at birth). Some of these are caused by a recognized single-gene disorder, chromosome aberration, or exposure to a teratogen; but most cases are of unknown origin and can be classified as multifactorial. Many chemicals, including many drugs, are known or suspected teratogens.

611

32.4.1

Biochemistry and Genetics

32.4.1. Nongenetic Causes Of Congenital Malformations Teratogenic agent a) Maternal conditions Advanced age Diabetes Phenylketonuria High fever Systemic lupus erythematosus b) Intrauterine infections Rubella Cytomegalovirus Toxoplasmosis Syphilis c) Chemicals Alcohol Phenytoin Valproic acid Cocaine Amphetamine Retinoic acid Lithium Mercury Warfarin Thalidomide d) Radiation e) Cigarette smoke

Typical malformations Cardiovascular and CNS malformations Microcephaly, mental retardation Microphthalmia, microcephaly, neural tube defects

Cataracts, deafness, patent ductus Various malformations in 5 % of infected pregnancies Sabre shin, Hutchinson teeth etc. Mental deficiency, growth retardation, facial abnormalities, joint anomalies, heart defects Craniofacial malformations, growth retardation, limb defects, mental deficiency Neural tube defects, heart defects Cerebral infarction, low birth weight, microcephaly Ear, brain and heart malformations Chondrodysplasia Phocomelia (a limb reduction defect) Microcephaly, ocular defects Low birth weight, incr. rate spontaneous abortion

Most teratogens act only during a specific period of gestation (examples: rubella before 16 weeks, thalidomide 20–36 d post-conception). But “fetal alcohol syndrome” can be caused by alcohol consumption at any time during pregnancy. In general, the most vulnerable period is in the first trimester. This is the time when most pregnant women develop pregnancy sickness, with nausea and food aversions. Pregnancy sickness is considered a protective

612

d) e)

Mercury Warfarin Thalidomide Radiation Cigarette smoke

Chondrodysplasia Phocomelia (a limb reduction defect) Microcephaly, ocular defects Low birth weight, incr. rate spontaneous abortion

Nongenetic Causes Of Congenital Malformations

32.4.1

Most teratogens act only during a specific period of gestation (examples: rubella before 16 weeks, thalidomide 20-36 days post-conception). But “fetal alcohol syndrome” can be caused by alcohol consumption at any time during pregnancy.that In general, vulnerableofperiod is in the first trimester. This is mechanism limitsthe themost ingestion food-borne teratogens. the time when most pregnant women develop pregnancy sickness, with nausea food aversions. Pregnancy sickness is considered a protective mechanism Theand multifactorial malformations are threshold traits, with a clear distinction between northat limits the ingestion of food-borne teratogens. mal and abnormal phenotype. Empiric can be applied. Weclear assume that the “liability” The multifactorial malformations arerisks threshold traits, with a distinction and abnormal phenotype. Empiric risks but can be for the defectbetween shows normal continuous variation in the population, only individuals beyond a applied. We assume that the “liability” for the defect shows continuous variation certain show defect:beyond a certain threshold show the defect: in thethreshold populaiton, but onlythe individuals

Threshold Frequency

Liability

Cleft lip and palate Some cases 273 are caused by single-gene disorders, chromosome aberrations, teratogens, or rubella, but most are multifactorial. Cleft palate is genetically different from cleft lip (with or without cleft palate). Risk of cleft lip: • 0.1 % total population • 4 % sibs of one affected child • 9 % sibs of two affected children • 30 % concordance in monozygotic twins The empiric risk increases with increased severity of the defect in affected family members. Pyloric stenosis Abnormal narrowing of the pylorus is caused by hyperplasia of the pyloric muscle. This results in propulsive vomiting. Surgical correction is often required. Frequency: 0.55 in males, 0.1 % in females Recurrence risk: Higher in relatives of affected females than in relatives of affected males.

613

32.5

Biochemistry and Genetics

According to the multifactorial threshold model, the risk is higher for close relatives of the less commonly affected sex than for close relatives of the more commonly affected sex. Neural tube defects Anencephaly is agenesis of the midbrain and forebrain, with death shortly before or after birth. Spina bifida is failure of fusion of the vertebral arches in the lower part of the spinal column. In severe cases, there are protruding meninges (meningocele) or meninges + neural elements (meningomyelocele). This is an important cause of paraplegia! Incidence: About 1:500 in North America Cause: Some cases have a specific cause (teratogens, trisomy 13), but the large majority are multifactorial. Recurrence risk after the birth of an affected child: 4 %. A previous child with anencephaly means an increased risk not only for anencephaly but also for spinal bifida in a next child, and vice versa. Environmental factors: Periconceptional folic acid reduces the risk of neural tube defects. Because in the US, one half of all pregnancies are unplanned and folic acid supplementation at the time an unplanned pregnancy can will detected comes too late, the current recommendation is 400 µg folic acid/day for all women of reproductive age. Prenatal diagnosis: Ultrasound, screening of maternal serum α-fetoprotein, followed by determination of α-fetoprotein in amniotic fluid obtained with amniocentesis. Heart defects Types: There are many types of congenital heart defect: septal defects, aortic stenosis, pulmonary stenosis, patent ductus, tetralogy... Incidence: 1 in 150 overall, with all degrees of severity from minimal impairment to lethal. Causes: Sometimes a cause can be found, but 80 % are “multifactorial”. The known causes include several single gene disorders, contiguous gene disorders like DiGeorge syndrome, as well as infections. Recurrence risk: For individual defects, the recurrence risk after the birth of an affected child is close to the square root of the population incidence. The risk is increased if two family members are affected. For heart defects overall, the recurrence risk after the birth of an affected child is 2 %, for the child of an affected parent 4 %, and 10 % if two first-degree relatives are affected.

614

Quantitative Trait Loci (QTLs)

32.5

32.5. Quantitative Trait Loci (QTLs) The identification of QTLs for continuously variable traits or for multifactorial disorders (“susceptibility genes”) is a major aim of current genetic research. The possibilities are: 1. A common polymorphism modifies the disease risk. Such polymorphisms can be maintained by balancing selection, either through simple heterozygote advantage or because the disease-promoting allele does something useful for those who carry it without developing the disease. 2. Disease susceptibility is caused by the accumulation of mildly deleterious mutations. Although a single mutation is insufficient to cause the disease, a constellation of several mutations along with an unfavorable environment puts people at risk. The mutations do nothing useful and can be considered “genetic garbage”. It is estimated that the average child is born with 2 or 3 mildly unfavorable new mutations on top of those inherited from the parents. 3. The disease is caused by a single major gene in a small subpopulation of patients. Also normal variation for traits such as blood pressure and intelligence may be due either to common polymorphisms that are maintained by balancing selection, or rare gene defects that are continuously created by mutation and removed by selection. A susceptibility gene that is important in one population may be unimportant in another. Example: A polymorphic aldehyde dehydrogenase is an important genetic determinant of alcoholism in Orientals, but not in Westerners. For this reason, geneticists searching for QTLs like to work with genetically homogeneous populations in places such as Iceland or Finland. Methods: Linkage studies These studies require families with multiple affected members. To make a genome-wide scan, a large number (> 100) of highly polymorphic VNTRs have to be used. In one popular design, affected sib pairs are studied under the assumption that there is more than the otherwise expected 50 % sharing of alleles near the QTLs. With hundreds of VNTRs, there is always a high chance that one of them reaches a moderately high Lod score even in the complete absence of linkage. Therefore only Lod scores higher than 4 or 5 are strong evidence for linkage. A genome map, but further gene mapping is needed to identify the gene itself. Extremely discordant sib pairs can be used to find genes for continuously variable traits. Linkage studies are very costly, and they require sophisticated statistical methods. Also, undisclosed nonpaternity can bias the results, especially in studies of extremely discordant sibpairs.

615

32.6

Biochemistry and Genetics

Candidate genes In some multifactorial conditions, known genes are suspected to be involved, for example genes for HLA antigens in autoimmune diseases. You have to know the genetic polymorphism first. Then you determine the allele frequencies in a group of patients and a matched control group. If an allele is over-represented in the patients, it is a bona-fide risk factor for the disease. This method is easier and more powerful than linkage studies, but it cannot detect “unexpected” susceptibility genes. This method is known as an association study. As with all epidemiological methods, choosing a control group that is truly matching is the most important and difficult step. single-nucleotide polymorphism (SNP) account for the majority of sequence variants in our genome. There are a few million SNPs, at least 100 000 of them in the coding and regulatory sequences of genes. They will be used for association studies on a genome-wide basis, using DNA chips to test for thousands of them at a time. Problem: An SNP variant that is associated with a particular condition (such as asthma, hypertension, or high intelligence) may either be the cause of the trait, or it may be in linkage disequilibrium with another polymorphism that causes the trait. Association does not prove causation! Linkage disequilibrium is defined as the occurrence together on the same chromosome of specific alleles at two closely linked loci more often than would be expected from the allele frequencies of the alleles involved.

32.6. Examples Of Susceptibility Genes Type I diabetes is associated with the DR3 and DR4 alleles in the major histocompatibility complex (the genes encoding the HLA antigens). An allele of the HLA-DQ gene reduces the risk of type I diabetes one hundred-fold. Also most other autoimmune diseases, such as ankylosing spondylitis, multiple sclerosis, and autoimmune thyroiditis, are associated with HLA antigens. In addition to HLA antigens, variations at a closely linked VNTR near the proinsulin locus contribute to the disease risk for type I diabetes. Type II diabetes is usually multifactorial; some chromosomal regions but no genes have been implicated in this, the more common type of diabetes. The heritability of this subtype is higher than in type I diabetes. One subtype, “maturity onset diabetes of the young” (MODY), is caused by a dominant gene (glucokinase deficiency in some patients; 6 different genes identified). This entity should probably be known as diabetes mellitus type III. Alzheimer’s disease is associated both with common polymorphisms and some rare mutations. The disease is more likely, and begins earlier, in individuals carrying the

616

Objectives in Brief

32.7

apolipoprotein E4, variant. This apoE variant is deposited along with β-amyloid in senile plaques. Some other genomic locations have been pinpointed in linkage studies. In a few families with early-onset Alzheimer (< 60 a), the disease is caused by a dominantly expressed point mutation in the gene for amyloid precursor protein (APP), the precursor of β-amyloid. Mutations in at least two other genes are known to cause early-onset Alzheimer. The APP gene is positioned on chromosome 21, and overexpression due to the extra gene copy is probably the reason why Down syndrome patients often develop Alzheimer. Osteoporosis is related to a common polymorphism in the gene for the calcitriol (vitamin D) receptor. Asthma risk is affected by a polymorphism in the gene for a protein in bronchial mucus (Clara cell secretory protein, CC16). In addition, several chromosomal regions have been identified by linkage. Atherosclerosis is usually multifactorial with rather low heritability (bad habits are more important than bad genes). But some patients have a single-gene defect, for example familial hypercholesterolemia (defective LDL receptor); high level of Lp(a); or “familial combined hyperlipoproteinemia” (presumably causing increased synthesis of apoB100).

32.7. Objectives in Brief 1. Explain why multifactorial traits usually show a bell-shaped phenotypic distribution. 2. Define the term “heritability” and state its limitations for analyzing the nature-nurture problem. 3. Make semi-quantitative statements about the hereditability of some multifactorial traits, including height, body weight, life-span, athletic performance, intelligence, alcoholism and criminal behavior. 4. State the approximate population incidence of multifactorial conditions including Type I and Type II diabetes, schizophrenia, manic-depressive disorder, cleft lip, neural tube defects and congenital heart defects, and the approximate incidence of these disorders in first degree relatives of a patient. 5. Specify the typical effects of the number of affected relatives and of the severity of the disease in an affected relative on the risk of multifactorial disorders. 6. State the roles of teratogens and intrauterine infections in the pathogenesis of congenital malformations.

617

32.7

Biochemistry and Genetics

7. State which methods can be used for the prenatal diagnosis of congenital malformations. 8. Apply the multifactorial threshold model to predict the relative risk to first-degree relatives of patients with a sex-influenced multifactorial disorder. 9. Describe the importance of identifiable single-gene effects in multifactorial disorders including autoimmune diseases, types I and II diabetes. 10. Describe the effect that major gene and minor gene alleles have for the risk assessment in diseases where they occur, and state examples of each in Hirschsprung’s and Alzheimer disease. 11. State the possible impact of pre-symptomatic testing for susceptibility genes for multifactorial disorders on disease incidence and on the use of prenatal diagnosis with elective abortion.

618

Part VII.

Appendix

33. Answers to the example questions 33.1. Thermodynamics 1) Entropy, driving force of a reaction • ∆G = ∆H − T × ∆S • Boiling point: Equilibrium liquid/gas → ∆G = 0 J/mol • 0 J/mol = 40 700 J/mol − 373.14 K × ∆S • 373.14 K × ∆S = 40 700 J/mol • ∆S =

40 700 J mol−1 373.14 K

≈ 100 J mol−1 K−1

2) Ligand binding to receptor X + R * ) RX

([R]t − [RX])[X] [R][X] = | ×[RX] [RX] [RX] Kd [RX] = ([R]t − [RX])[X] | : ([R]t − [RX]) Kd [RX] [X] = ([R]t − [RX]) 1nM × 1000 1 1 = = nM = nM 10000 − 1000 10 − 1 9 ≈ 0.11nM Kd =

3) Virus capsid stability The high activation energy of the dissociation makes virus metastable. Note that most of the other options would actually decrease virus stability, rather than increasing it. The difference between medicine and shamanism or quackery is the belief that the rules inside a living organism are the same as those in the test tube and that therefore disease can be cured by applications of the laws of science.

621

33.1

Biochemistry and Genetics

4) Elimination of drugs from the body Since the blood sample was taken 3 half life periods after injection, the concentration after injection must have been 2 × 2 × 2 = 8 times that found in the experiment, hence 8 × 12.5 nM = 100 nM. The amount of the drug is concentration times volume, hence 100 nmol/l × 50 l distribution volume = 5000 nmol = 5 µmol. Since 1000 g : 1 mol = x g : 5 µmol. Separating known and unknown variables yields x = 1 × 103 g × 5 × 10−6 mol : 1 mol = 5 × 10−3 g = 5 mg.

pKa GG B + H+ 5) Effect of pKa on membrane diffusion BH+ E GGGGGGGGC The higher the pKa is above the environmental pH (here blood pH, 7.4), the less of the drug is in the uncharged form which can cross the membrane. According to Fick’s diffusion law, this lowers transport velocity. The Henderson-Hasselbalch-equation allows us to calculate the ratio of charged over   [BH + ] = pKa − pH blood pH = 7.4 uncharged drug at the given pH: log [B]

Compound

pKa

[BH + ] [B]

Lidocaine Bupivacaine Procaine

7.7 8.1 8.9

2.00 5.01 15.84

6) Follow-up: Bacterial metabolism inside an abscess leads to the formation of lactic acid, which drops the pH. As a consequence lidocain can no longer enter the nerve cells, exposing the patient to a considerable amount of pain.

7) pH-dependent drug trapping :

622

33.2

Proteins

log

[HA] [A- ] [H + ]

= pK a - pH = 3.4 - 7.4 = -4

[HA] = 10 -4 [A- ] [H + ]

1 HA

10 000 H ++ A-

Plasma, pH = 7.4 Ratio =

Plasma membrane

10 001 1.01

= 9902

Stomach juice, pH = 1.4

log

[HA] [A- ] [H + ]

HA

H ++ A-

1

0.01

= pK a - pH = 3.4 - 1.4 = 2

[HA] = 10 2 [A- ] [H + ]

33.2. Proteins pKa GGGGGGGGB 1) Effect of pH on enzyme activity HA F GG H+ + A− Glu-35 (pKa = 5.9): pH ≤ pKa : more HA → higher activity Asp-52 (pKa = 4.5): pH ≤ pKa : more HA → lower activity 2) Structure of alpha-amino acids, radioactivity A β-radiation is emission of electrons by decay of a neutron of the nucleus into a proton and an electron. B Decarboxylation of an amino acid produces CO2 from the carboxy-group. Since that C-atom is radioactive, the CO2 will also be. C The carboxy-group is attached to the rest of the molecule by a very stable c−C-bond. Sparging with radioactive CO2 will therefore not exchange this group. D For most intents and purposes, the physico-chemical properties of radioactively labelled compounts are identical with the unlabelled ones. Indeed, on this identity their use in tracer studies rests.

623

33.2

Biochemistry and Genetics

E see above. 2.18 +G GGGGGGGB 3) pI-value of amino acid HOOC CHNH+ GG 3 CH2 NH3 F

− OOC

+ CHNH+ 3 CH2 NH3

8.95 − 10.53 + GGGGGGGGGB − GGGGGGGGB F GG OOC CHNH2 CH2 NH3 F GG OOC CHNH2 CH2 NH2 1/2 × (8.95 + 10.53) = 1/2 × 19.48 = 9.74 4) Properties of amino acids At pH 8.0 Glu is negatively charged, while Val is not. A Glu→Val mutation will reduce movement towards the positive pole. At pH 1.0 Glu is uncharged, as is Val. Hence the mutation has no effect on electrophoretic mobility at this pH. 5) Functional replacement of amino acids Ala is Ser minus the −OH group which is the catalytically active part of Ser. 6) pH dependence of solubility of amino acids Glu + Asp acidic residues, give additional charges at alkaline pH. 7) Determination of protein concentration from UV-absorption Light absorption A as a function of concentration c and path length l is described by Lambert-Beer’s law   I A = − log I = λ c l, where λ is the extinction coefficient at wavelength λ. In our case 0 the sample was diluted 100-fold, as Lambert-Beer’s law is valid for most substances only if A ≤ 1.0. cm Hence the molar protein concentration c = 100× Al = 100× 4 ×0.410mol 4 ×1 l cm = λ 1.0 × 10−3 mol/l.

40 −4 mol/l 4 ×10

=

Since the average molecular weight is 65 kDa, and hence 65 × 103 g is equivalent to 1 mol, we can convert the molar concentration into a weight concentration: 1.0 × 10−3 mol/l × 65 × 103 g/mol = 1.0 × 65 g/l = 65 g/l = 6.5 g/dl, which is in the normal range. 8) Strange disease The description is typical for a case of FFI. 9) Collagen related inherited disease The case description is typical for chondrodysplasia, the molecular genetics confirms this.

624

Enzymes

33.3

10) CAG-length variation Huntington’s disease caused by CAG-expansion in huntingtin → poly-Glu amyloid formation

33.3. Enzymes 1) Enzyme turnover rate: There are two ways to answer this question, both are equally valid: the industrious student Vmax ∗ [S] (Km + [S]) V ∗ 3 Km = max (Km + 3 Km ) = Vmax ∗ 3/4

v=

(33.1) (33.2) (33.3)

the bright student no calculation required: • [S] > Km → v > 1/2 Vmax eliminates A–C • [S] < ∞ → v < Vmax eliminates E • D is the only possible option! 2) Enzyme reaction velocity When the substrate concentration is (nearly) saturating, v ≈ Vmax . However, Vmax = kcat × [E], thus doubling [E] doubles the velocity. 3) Enzyme turnover number First Calculate molar amount of enzyme 1 mol × 5 × 10−6 g 5 × 104 g = 10−6 × 10−4 mol

x=

= 1 × 10−10 mol Now turnover number 1 × 10−5 mol/min 1 × 10−10 mol = 10−5 × 1010 min−1

kcat =

= 1 × 105 min−1

625

33.4

Biochemistry and Genetics

4) Enzyme activity, HMM-equation An enzyme with the higher activity will turn over more substrate. An enzyme with lower Km can do so even at low [S]. 5) Action of pharmaceutical When interpreting plots always look at the labels of the axis first.This is a time-dependent inactivation by a suicide substrate. Inhibitions are reversible, diffusion controlled reactions that happen on a ms time scale. 6) Metabolic and thermodynamic direction Le Chatelier’s principle: removal of the product stimulates its formation. 7) Henri-Michaelis-Menten law, Lineweaver-Burk plots The parallel lines indicate an uncompetitive inhibition. To get Km we locate the line in the absence of inhibitor (red). Its x-intercept (−1/Km ) is −0.5 mM−1 , thus Km = 2 mM. 8) Hill-equation, co-operative binding θmax × [O2 ]h K0.5 + [O2 ]h 100% × 12.9 = 13 + 12.9 100% = 14 ≈ 7%

θ=

9) difference between inhibition and inactivation Covalent modification usually results in irreversible loss of activity → inactivation 10) Enzyme nomenclature, enzyme classes Transfer of a phosphate group from ATP to glycerol is catalyzed by a transferase, specifically a phosphotransferase or kinase.

33.4. Amino acid metabolism 1. Protein consumption • 56 g/d recommended - 34 g/d plant protein uptake = 25 g/d additional plant protein required

626

Digestion

33.5

• 75 g/d animal protein x 41 Mt / 7 Mt = 439.3 g/d of plant protein equivalent • - 25 g/d additional plant protein = 439.3 g/d wasted per US citizen • / 56 g/d recommended uptake = 7.4 daily protein doses wasted per US citizen • x 3 × 108 US citizens = 2.2 × 109 daily doses • That is about 1/3 of the worlds population!

2. Biological value of proteins The ratio of the mixture 2 parts rice, 1 part soy is calculated as weighted average of the ratios for the individual food stuffs, weighting factor is the proportion in the mixture. For Ile that yields: 2 × 4.08 + 1 × 3.15 = 3.46 2+1 amino acid

ratio rice

ratio soy

(33.4)

ratio mix

Ile 3.15 4.08 3.46 Leu 4.41 3.95 4.26 Lys 2.67 4.43 3.26 Met 1.58 0.69 1.28 Phe 2.72 2.51 2.65 Thr 4.29 4.29 4.29 Trp 0.67 3.33 1.56 Val 4.33 3.56 4.07 After filling the table for all amino acids you see that Met is now limiting, with a ratio of 1.28. This determines the biological value of the protein. Note that while both rice and soy proteins individually lack certain essential amino acids (ratio < 1.0), the mixture provides all of them in super-optimal amounts, that is, has a high biological value. Such calculations are important for making food mixtures for parenteral nutrition.

33.5. Digestion 1) Weight loss on 0 J diet: Under those circumstances, patient would not be very active, energy consumption would be limited to resting metabolism, on average 7500 kJ/d in |, 5400 kJ/d in ~. Fat yields 38.9 kJ/g of energy when it is metabolized. So | would loose just under 200 g/d, ~ just under 140 g/d, if only body fat were metabolized.

627

33.6

Biochemistry and Genetics

2) Loss of lean muscle mass during diets: To maintain a constant blood [glucose] the body needs to perform gluconeogenesis. Fatty acids can not be used for that (why?), glycerol from fat catabolism can, but amount is insufficient. Amino acids from muscle protein is used instead. 3) Brain glucose metabolism during fasting: The brain uses energy at a fairly constant rate, irrespective of the metabolic situation of the body and of intellectual activity. Therefore, brain glucose consumption is not hormone regulated. As we will see later (see chapter 31 on page 569), in long term fasting ketone body concentrations in blood become high enough to supply part of the energy the brain needs, sparing glucose and hence lean muscle mass. 4) Athlete in distress: The patient has used up his entire glycogen stores during his strenuous activity. The only way to maintain his blood glucose level is gluconeogenesis, which however is inhibited by alcohol. Thus hypoglycemia results in loss of consciousness, and has to be corrected by infusion of glucose to save his life. Note that Ringer/Lactate would not work under these conditions! (Why?)

33.6. Integration of metabolism 1) Wasting in terminal illness: Glucocorticoids like cortisol are released as consequence of chronic stress. 2) Obesity: Low levels of leptin may cause obesity, while high levels may be the result. Both reduced sirtuin activity and changes in B/F ratio in the gut are regularly observed in obesity. However, amenorrhea in ~ is more a consequence of anorexia (albeit it may occur secondary to long-term damage in severe obesity). 3) Metabolic syndrome: In a patient with increased hip circumference one would expect a reduction of HDL (good cholesterol), not an increase.

628

34. Tables

629

34.3

Biochemistry and Genetics

34.1. Conversion from non-metric to metric units

non-metric 1 cal 1 cup 1 inch 1 lb 1 oz 1 tablespoon 1 teaspoon

metric 4.1868 J 236.6 ml 2.54 cm 453.59 g 28.350 g 15 ml 5 ml

34.2. Symbols used

C q T

630

heat capacity heat Temperature

W/K K or °C

Greek alphabet

34.3

34.3. Greek alphabet alpha beta gamma delta epsilon zeta eta theta iota kappa lambda mu nu xi o pi rho sigma tau upsilon phi chi psi omega

α β γ δ , ε ζ η θ, ϑ ι κ λ µ ν ξ o π, $ ρ, % σ, ς τ υ φ, ϕ χ ψ ω

A B Γ ∆ E Z H Θ I K Λ M N Ξ O Π R Σ T Υ Φ X Ψ Ω

631

34.5

Biochemistry and Genetics

34.4. The genetic code

mRNA-codons and the corresponding amino acids. Alternative uses of codons are marked on the exterior. The colors used to symbolize different compounds are known as “Shapely color set”, a quasi-standard in molecular modeling. Figure from [Buxbaum, 2007].

632

The genetic code

34.5

633

34.5

Biochemistry and Genetics

34.5. Periodic system of the elements

634

35. Acronyms AAV adeno-associated virus, used in gene therapy ABC ATP-binding cassette, largest class of primary active trans-membrane transporters ACAT Acyl-CoA-cholesterol-acyl transferase ACE angiotensin converting enzyme, involved in blood pressure regulation ACP Acid phosphatase, enzyme elevated in late stages of prostrate cancer ACTH adrenocorticotrophic hormone ADA adenosine desaminase ADH alcohol dehydrogenase, enzyme found mostly in liver and involved in ethanol detoxification ADHS attention deficit hyperactivity syndrome, disease in children of controversial existence, causation and treatment ADP adenosine diphosphate AGE advanced glycation end-products, formed from proteins and aldoses (esp. glucose) AGT aspartate:glutamate aminotransferase, liver enzyme AI adequate intake, dose recommendation for nutrients in those cases where scientific evidence is insufficient to set RDA-values AICD APP intracellular domain, cytosolic fragment of APP produced by secretases AID artificial insemination by donor AIDS acquired immuno-deficiency syndrome, disease cause by infection with a retro-virus, HIV Akt AKR mouse directly transforming retrovirus associated oncogene, also known as protein kinase B ALA δ-aminolevulinate, intermediate of heme synthesis ALE advanced lipoxydation endproducts, formed from proteins and lipoperoxides

635

35

Biochemistry and Genetics

ALP Alkaline phosphatase, enzyme that appears in serum after bone and liver damage ALT Alanine transaminase, enzyme that appears in serum after liver damage AMP adenosine monophosphate AMPA 3-amino-3-hydroxy-5-methyl-4-isoxacol propionate, agonist of certain glutamatereceptors ANF Atrial natriuretic factor APC adenomatous polyposis coli gene, gene involved in colon cancer formation apoA apolipoprotein A, major apolipoproteins of HDL apoB apolipoprotein B apoC apolipoprotein C apoE apolipoprotein E APP β-amyloid precursor protein, involved in plaque formation in Alzheimers disease APTT Activated partial thromboplastin time ARB angiotensin receptor blocker, class of pharmaceuticals used to lower blood pressure and manage diabetic nephropathy ASO application specific oligo, DNA segment used as probe on gene chips AST aspartate transaminase, enzyme that appears in serum after liver damage ATM ataxia telangiectasia mutated kinase ATP adenosine triphosphate BARK β-adrenergic receptor kinase BioH2 dihydrobiopterine BioH4 tetrahydrobiopterine BMI body mass index, calculated by dividing body weight (kg) by the square of the height (m), should be 18–25 BMR basal metabolic rate, energy expenditure in the absence of activity BPG 2,3-Bisphosphoglycerate, regulator of oxygen affinity of hemoglobin BRCA breast cancer, genes that increase the likelihood of breast cancer development CAM cell adhesion molecule CATH class architecture topology homology at http://www.cathdb.info/latest/index.html

636

Acronyms

35

cAMP cyclic AMP, second messenger CBB Coomassie brilliant blue, group of dyes used to stain proteins in gels Cdk cyclin dependent protein kinase cDNA complementary DNA, DNA generated by action of a reverse transcriptase upon a mRNA CDP cytidine diphosphate CF cystic fibrosis, inherited disease cGMP cyclic GMP, second messenger CETP cholesterol ester transfer protein CFTR cystic fibrosis transmembrane regulator, protein defect in cystic fibrosis CHD coronary heart disease CHO carbohydrates CJD Creutzfeldt-Jakob-disease, a prion disease CK creatine kinase, enzyme which appears in serum after damage to muscle, heart or brain. Source can be determined by iso-enzyme analysis cMOAT canalicular multiple organic anion transporter, defect in Dubin-Johnson-syndrome CNS central nervous system, brain and spinal cord CoA coenzyme A, carrier of acyl residues in metabolism CRABP cellular retinoic acid binding protein, receptor for retinoic acid CRBP cellular retinol binding protein, receptor for retinol Cre , recombinase used in the production of knock-out and knock-in mice CREB cAMP response element binding, family of transcription factors regulated by cAMP CRP C-reactive protein, acute-phase serum protein CSF cerebrospinal fluid, fluid surrounding the CNS and separated from serum by the blood/brain barrier CTAB cetyl trimetylammonium bromide, positively charged (cationic) detergent CTL cytotoxic lymphocyte CTP cytidine triphosphate dATP deoxy-ATP, used for making DNA

637

35

Biochemistry and Genetics

DCCD dicyclohexylcarbodiimide, substance used to activate carboxy-groups in chemical synthesis dCTP deoxy-CTP dGTP deoxy-GTP DHA docosahexaenoic acid, polyunsaturated fatty acid, possibly a vitamin DHF dihydrofolate DHFR dihydrofolate reductase DMD Duchenne muscular dystrophy DNA desoxyribonucleic acid l -DOPA 3,4-dihydroxy phenylalanine DRI Dietary reference intake, EAR, RDA and UL of a nutrient DTPA , chelator used against iron poisoning dTTP deoxy-Thymidine triphosphate EAR estimated average requirement, dose of a nutrient required by an average member of the group studied EDTA ethylene diamine tetraacetic acid, chelator EGF Epidermal Growth Factor ELISA enzyme linked immunosorbent assay, method to determine the concentration of an antigen or antibody env envelope, transmembrane proteins in the envelope of virus, required for binding to the target cell EPA eicosapentaenoic acid, polyunsaturated fatty acid, possibly a vitamin EPO erythropoietin, hormone produced by the kidney that stimulates erythrocyte formation in bone marrow ER endoplasmic reticulum, intracellular membrane system ERAD ER associated protein destruction, mechanism that destroys incorrectly folded proteins during their maturation in the ER erb ? Erk extracellular signal regulated kinase, protein in the MAP-kinase pathway ES embryonic stem cells, pluripotent stem cells extracted from embryos

638

Acronyms

35

EST expressed sequence tags, short pieces of cDNA that identify genes expressed in a cell or tissue FAD flavin adenine dinucleotide, oxidized FADH2 flavin adenine dinucleotide, reduced, cofactor of flavo-proteins FFI fatal familial insomnia, a prion disease FGF Fibroblast Growth Factor FIGLU N-Formiminoglutamate, intermediate in His-breakdown FISH fluorescent in-situ hybridization, method to localize genetic information on microscopic slides Flip , recombinase recognizing the FRT DNA sequence. Used in a similar fashion as Cre/LoxP FMN flavin mononucleotide, cofactor in some oxidoreductases Fmoc N-α-9-fluorenylmethoxycarbonyl, protective group during Merrifield protein synthesis fos Finkel-Biskis-Jinkins osteosarcoma oncogene FRT , DNA element recognized by the Flip recombinase FSH follicle stimulating hormone FSSP families of structurally similar proteins at http://www.chem.admu.edu.ph/ nina/rosby/fssp.htm G6PDH glucose-6-phosphate dehydrogenase, enzyme of the pentose phosphate pathway, defective in favism GABA γ-amino butyric acid, a neurotransmitter gag group-specific antigen, protein in retro-virus capsid Gal galactose, a hexose GDP guanosine diphosphate GFR glomerular filtration rate, volume of blood filtered by the glomerulus per unit time GFP green fluorescent protein, marker in molecular biology studies GI gastrointestinal Glc glucose, a hexose, essential component of food GlcNAc N-acetylglucosamine, sugar derivative found in glycoproteins and -lipids GM-CSF granulocyte/macrophage colony stimulating factor, cytokine

639

35

Biochemistry and Genetics

GMP guanosine monophosphate GOT glutamate:oxoglutarate transaminase, liver enzyme GPI glycosyl phosphatidylinositol GPT glutamate:pyruvate transaminase, liver enzyme GSS Gerstmann-Stäussler-Scheinker-disease, a prion disease GTP guanosine triphosphate GuHCl guanidinium hydrochloride, substance that destroys protein secondary, tertiary and quaternary structure HbA adult hemoglobin,(α2 β2 ) HbF fetal hemoglobin,(α2 γ2 ) HbS sickle cell hemoglobin, Glu-6→Val in β HCG human chorionic gonadotropin HDL high density lipoprotein, “good cholesterol” HDN late-onset haemorrhagic disease of the newborn, treated with vitamin K HFE hemochromatosis HGPRT hypoxanthine:guanine phosphoribosyl transferase HIC hydrophobic interaction chromatography, method to separate proteins HIF hypoxia inducible transcription factor, transcription factors which regulate gene expression depending on available oxygen HIV Human immuno-deficiency virus, causative agent for AIDS HLA Hauptlymphozyten-Antigen, synonym for MHC HMG hydroxymethyl glutaryl-, also: human menopausal gonadotropin HMM Henri-Michaelis-Menten HPLC high performance liquid chromatography, chromatography on fine-grained, homogenous stationary phases to increase separation power HRE hypoxia response element, segments in the promotor of a gene, which bind HIF Hsc70 70 kDa heat shock cognate, constitutively expressed isoform of Hsp70 Hsp heat shock protein, group of chaperons or chaperonins amplified under stress conditions ICSI Intracytoplasmic sperm injection

640

Acronyms

35

IDL intermediate density lipoprotein IDoM inherited disease of metabolism IEC ion exchange chromatography, method to purify proteins based on their charge IEF isoelectric focussing, method to separate proteins based on their different pI IFN interferon Ig immunoglobulin IgA immuno-globulin A IgD immuno-globulin D IgE immuno-globulin E IGF Insulin-like Growth Factor IgG immuno-globulin G IgM immuno-globulin M IMP inosine monophosphate INH isonicotinic acid hydrazide, isoniacid, tuberculostaticum INK4 inhibitor of kinase 4 IP3 inositol-1,4,5-trisphosphate, second messenger IQ intelligence quotient, result of an intelligence test IUBMB International Union for Biochemistry and Molecular Biology IUPAC International Union of Pure and Applied Chemistry IVF In-vitro fertilization jun Junin virus oncoprotein lac lactose LWB low weight at birth LCAT lecithin-cholesterol acyl transferase LDH Lactate dehydrogenase, enzyme which appears in serum after tissue damage. Source can be determined by iso-enzyme analysis LDL low density lipoprotein, “bad cholesterol” LoxP , DNA element recognized by the Cre recombinase

641

35

Biochemistry and Genetics

LPL lipoprotein lipase, enzyme that cleaves triglycerides LH luteinizing hormone LT leukotriene MAO monoamine oxidase MAP mitogen activated protein [MCAD medium chain acyl-CoA dehydrogenase, enzyme in fatty acid catabolism MCD multiple carboxylase deficiency, inherited disease Mdm Murine double minute, oncogene MEK MAP kinase/Erk kinase, protein in the MAP pathway MHC major histocompatibility complex mRNA messenger ribonucleic acid, RNA that gets translated into proteins MS mass spectrometry, sensitive analytical technique that identifies molecules by molecular weight MSUD maple syrup urine disease, inborn error of amino acid metabolism MTX Methotrexate, dihydrofolate reductase antagonist used against cancer and autoimmune diseases MW molecular weight (more correctly: -mass) myc myelocytomatosis proto-oncogene NAD+ nicotinamide adenine dinucleotide, oxidized NADH + H+ nicotinamide adenine dinucleotide, reduced, soluble carrier of activated hydrogen in catabolic reactions NADP+ nicotinamide adenine dinucleotide phosphate, oxidized NADPH + H+ nicotinamide adenine dinucleotide phosphate, reduced, soluble carrier of activated hydrogen in anabolic reactions NAD(P)H either NADH or NADPH N.C.E.P. National cholesterol education program neor neomycin resistance, genetic marker used in DNA technology neu ? NF-κB nuclear factor κB

642

Acronyms

35

NGF Nerve Growth Factor NMDA N-methyl-D-aspartate, agonist of inotropic glutamate receptors in the nervous system NMR nuclear magnetic resonance, method to determine the 3D-structure of molecules NO· nitric oxide, paracrine hormone NPU net protein utilization, measures biological value of proteins NPY neuropeptide Y

OMIM online Mendelian inheritance in man, catalogue of inherited diseases at http://www.ncbi.nlm.nih.gov/sites OPA ortho-phtaldialdehyde, chemical that gives fluorescent compounds with primary amines PAGE polyacrylamide gel electrophorese, separation method for proteins and nucleic acids PAH phenylalanine hydroxylase, protein defect in PKU PCNA proliferating cell nuclear antigen PCR polymerase chain reaction, method to amplify a particular stretch of DNA PDB protein data base, database of protein structure at http://www.rcsb.org/pdb/home/home.do PDI protein disulphide isomerase, enzymes responsible for the formation of correct disulphide bonds in the ER PDGF Platelet-derived Growth Factor PEP phosphoenol pyruvate PEPCK phosphoenolpyruvate carboxykinase PER protein efficiency ratio, used to express the biological value of proteins PET positron emission tomography, non-invasive method to measure metabolic activity of an organ, especially the brain PI3 phosphatidylinositol trisphosphate PKA protein kinase A, mediator of cAMP responses PKC protein kinase C, mediator of Ca responses PKU phenylketonuria PLC phospholipase C pol RNA-dependent DNA-polymerase, reverse transcriptase of retro-virus pRb retinoblastoma protein

643

35

Biochemistry and Genetics

PrP prion protein, prion stands for proteinaceous infectious agent PRPP phosphoribosyl pyrophosphate PTH parathyroid hormone PUFA poly-unsaturated fatty acids, fatty acids with several double bonds QTL Quantitative trait locus RA rheumatoid arthritis RAGE receptor for advanced glycation end-products of proteins Raf receptor associated factor, protein in the MAP-kinase pathway Ras rat sarcoma viral antigen, protein in the MAP-kinase pathway RBC red blood cell, erythrocyte RDA recommended dietary allowance RFLP restriction fragment length polymorphism RIA radioimmuno assay, method to determine the concentration of an antigen or antibody, largely superseded by ELISA RNA ribonucleic acid RNAi RNA interference, technique used to prevent expression of a specific gene product RISC RNA-induced silencing complex, involved in RNAi ROS reactive oxygen species, compounds derived from partially reduced oxygen molecules rRNA ribosomal ribonucleic acid, structural component of ribosomes with ribozyme activity RPC reversed phase chromatography, separation method for small organic molecules RQ respiratory quotient, ratio CO2 produced by O2 consumed SAH S-adenosyl homocysteine, demethylation product of SAM SAM S-adenosyl methionine, carrier of C1-bodies in metabolism SCOP structural classification of proteins at http://scop.mrc-lmb.cam.ac.uk/scop/ SCID severe combined immunodeficiency disease, inherited immuno-deficiency SDH succinate dehydrogenase, enzyme of Krebs-cycle SDS sodium dodecylsulphate, detergent used to destroy tertiary and quaternary structure in proteins

644

Acronyms

35

SEC size exclusion chromatography, synonym for gel filtration SLE systemic lupus erythematosus, autoimmune disease sis simian sarcoma, oncogene SNP single-nucleotide polymorphism snurp small nuclear ribonucleoprotein src Rous sarcoma virus oncogene STAT signal transducer and activator of transcription SUMO small ubiquitin-like modifiers, peptides transferred to Lys -amino groups in proteins for regulatory purposes TAP transporter associated with antigen presentation, transport antigenic peptides into the ER, encoded by the ABCB2 and ABCB3 gene Taq Thermus aquaticus, thermophile archaebacterium found in deep-sea hydrothermal vents (“black smokers”) TCA tricarboxylic acid, summary name for the intermediates of the Krebs-cycle TCR T-cell receptor, protein that binds to the antigen-loaded MHC on antigen-presenting cells TGF Transforming Growth Factor THF 5,6,7,8-tetrahydrofolate, active form of the vitamin folic acid TIBC total iron binding capacity LMP latent infection membrane protein TNF tumor necrosis factor TOBEC total body electric conductivity, method to determine body fat content TPP thiamin pyrophosphate, active form of vitamin B1 tRNA transfer RNA TSH thyroid-stimulating hormone UDP uridine diphosphate UL tolerated upper intake level, amount of a substance that does not cause negative effects in the majority of the population even when taken regularly over a long time US United States (of America)

645

35

Biochemistry and Genetics

US-RDA US recommended daily allowance, short version of the RDA, not differentiated by gender nor age, and not regularly updated. UTP uridine triphosphate UV ultraviolet, light with wavelength shorter than 400 nm vCJD variant Creutzfeldt-Jakob-disease, “mad cow disease” VLDL very low density lipoprotein, transports cholesterol from the liver to the tissues VNTR variable number of tandem repeats vWF von Willebrand factor, protein involved in platelet activation waf ? WHO World Health Organization YAC yeast artificial chromosome, used to clone large segments of DNA

646

Bibliography S.M. Berget, C. Moore, and P.A. Sharp. Spliced segments at the 5’ terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. USA, 74:3171–3175, 1977. URL http://www.pnas. org/content/74/8/3171.full.pdf+html. E. Buxbaum. Introduction to protein structure and function. Springer, New York, 2007. ISBN 978-0-387-26352-6. E. Buxbaum. Biophysical chemistry of proteins: An introduction to laboratory methods. Springer, New York, in press. D. Doenecke, J. Koolman, G. Fuchs, and W. Gerok. Karlsons Biochemie und Pathobiochemie. Thieme, Stuttgart, 15th edition, 2005. ISBN 3-13-357815-4. A. Ghazalpour, S. Doss, X. Yang, J. Aten, E.M. Toomey, A. Van Nas, S. Wang, T.A. Drake, and A.J. Lusis. Toward a biological network for atherosclerosis. J. Lipid Res., 45: 1793–1805, 2004. URL http://www.jlr.org/cgi/reprint/R400006-JLR200v1.pdf. R.S. Gibson. Principles of Nutritional Assessment. Oxford University Press, Oxford, 2nd edition, 2005. ISBN 978-0-195-17169-3. E.C.M. Mariman. Epigenetic manifestations in diet-related diorders. J. Epigenetics Epigenomics, 1:232–239, 2008. C.C. Metges and C.A. Barth. Metabolic consequences of a high dietary-protein intake in adulthood: Assessment of the available evidence. J. Nutrition, 130:886–889, 2000. D.L. Nelson, M.M. Cox, and A.L. Lehninger. Principles of Biochemistry. W.H.Freeman, New York, 5th edition, 2008. ISBN 978-1-429-20892-5. S. O´Rahilly, I.S. Farooqi, G.S.H. Yeo, and B.G. Challis. Minireview: Human obesity— lessons from monogenic disorders. Endocrinology, 144:3757–3764, 2003. doi: 10.1210/en. 2003-0373. J.J. Otten, J.P. Hellwig, and L.D. Meyers, editors. Dietary reference intakes. Institute of Medicine of the National Academies, Washington, 2006. ISBN 0-309-10091-7.

647

Bibliography

K. Sacksteder, B. Biery, J. Morrell, B. Goodman, B. Geisbrecht, R. Cox, S. Gould, and M. Geraghty. Identification of the a-aminoadipic semialdehyde synthase gene, which is defective in familial hyperlysinemia. Am. J. Human Genetics, 66:1736–1743, 2000. M.E. Shiles, J.A. Olson, and M. Shike, editors. Modern Nutrition in Health and Disease vol 1-3. Lea & Febiger, Philadelphia, 10th edition, 2005. ISBN 978-0-781-74133-0. S.K. Sieberts and E.E. Schacht. Moving towards a system genetics view of disease. Mamm. Genome, 18:389–401, 2007. doi: 10.1007/s00335-007-9040-6. C. Vonrhein, G.J. Schlauderer, and G.E. Schulz. Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases. Structure, 3:483–490, 1995.

648

Index α2 -macroglobulin, 336, 343 γ-carboxy-glutamic acid, 298 Na+ /K+ -ATPase, 341 A/B toxin, 63 ab initio, 52 ABC-R transporter, 291 ABC-transporter, 534 abetalipoproteinemia, 261, 375 ABO-system, 219–220 abortion, 564, 612 acetal, 7 acetaminophen, 462 acetazolamide, 156 acetoacetate, 366 acetone, 366 in protein precipitation, 68 acetyl-CoA, 61 acetyl-CoA carboxylase, 367 acetylation, 61, 117 acetylcholine, 275–277 achondroplasia, 236, 258 acid, 9–10 maltase deficiency, 354 phosphatase, 209 acid maltase, 519 acidosis, 174, 435, 582 lactic, 201 acne, 287, 291 aconitase, 245 acridine, 114 acrodermatitis enteropathica, 338 actinomycin A, 99

acute fatty liver of pregnancy, 533 acute phase reactant, 206 acyl transferase, 369 acyl-CoA dehydrogenase, 533 deficiency, 367 Acyl-CoA-cholesterol-acyl transferase, 373 adaptation, 304 adenoassociated virus, 552 adenomatous polyposis coli, 191, 495 adenosine desaminase, 509 adenosine triphosphate, see ATP adenosyl S-adenosyl homocysteine, 422 S-adenosyl methionine, 422 adenovirus, 111, 552 adenylate cyclase, 274 adenylate kinase, 167 adenylation, 63 adhesion focal, 488 adiabatic, 16 adipocyte, 289, 296 adiponectin, 595 adipose tissue, 356, 363–364, 402, 576 hyperplasia, 381 hypertrophy, 381 ADP ribosylation, 275 adrenal, 289, 334, 342, 356, 393 adrenocortical, 494 adrenoleucodystrophy, 534 adrenomyeloneuropathy, 534 aflatoxin, 347

Index

AGE, 57, 601 aggregation protein, 55 aging, 57, 254 AIDS, 104, 190, 193, 336, 565 Akt, 490 alanine cycle, 435, 579 transaminase, 209 albinism, 263, 336 occulocutaneous, 425 albumin, 204–205, 208, 289, 301, 308, 334, 336, 364, 461 alcohol, 394, 398, 452–454, 471, 473, 476, 531, 611, 612 dehydrogenase, 453 alcoholism, 376 aldehyde, 12 dehydrogenase, 453 aldehyde oxidase, 342 aldolase, 354 deficiency, 354 aldolase B, 518 aldosterone, 277, 323, 393, 402 ALE, 601 aleuron, 300 alkali disease, 347 alkaline phosphatase, 209 alkalosis, 174 alkaptonuria, 427 allele, 233 allergy, 294, 313 allopurinol, 471 allostery, 200, 222, 448, 569 Alport-syndrome, 46 Alu-sequence, 110, 111 Alzheimer’s disease, 616 Alzheimer’s disease, 80–82, 125, 558, 611 Amadori-rearrangement, 59 Amanita phalloides, 116 α-amanitin, 116, 462 amaurotic idiocy, 369

650

amenorrhea, 123, 130 post-partum, 477 Ames test, 114 amide, 7 amine oxidase, 333 amino acid, 27–34 essential, 406 metabolism, 405–438 oxidase, 410 amino sugar, 358 aminoacyl-tRNA synthetase, 98 δ-aminolevulinate synthase, 457 aminophylline, 274, 276 ammonia, 10, 399 amphetamine, 612 amphipatic, 41, 371 amphophilic, 72 AMPylation, 63 amylase, 210, 316, 439, 442 amyloid, 74, 125, 617 serum, 595 Amytal, 252 anabolic, 447 analbuminemia, 205 anaplerotic reaction, 245 Anderson disease, 519 androgen, 130, 513 anemia, 201, 207, 296, 299, 302, 308, 311, 318, 334, 343 Heinz-body, 517 hemolytic, 469 iron deficiency, 479, 481 megaloblastic, 464 sickle cell, 226, 558 anencephaly, 337, 474, 614 aneuploidy, 123 Anfinsen-hypothesis, 52 Angelman syndrome, 268 angina, 208 angiotensin, 275, 276, 393 angiotensinogen, 595 anhydride, 7

Index

phospho-, 7 ankylosing spondylitis, 508 annealing, 93 anomer, 12 anoxia, 253 antabuse, 453 antacid, 321, 481 antagonist, 286 anti-sense, 550 anti-vitamin, 286 antibiotic, 99, 298, 300, 309, 311 antibody, see immunoglobulin catalytic, 163 anticipation, 235 anticodon, 98 anticonvulsant, 293, 311, 316, 317, 321, 336, 481 antimycin A, 252 antioxidant, 294, 355 antiport, 172 antitrypsin, 206 antivitamin, 303 aorta, 257 Apert syndrome, 258 Apicomplexa, 51 apoA I, 374, 375 apoB 48, 372 100, 372, 373, 376 apoE, 373, 374, 376, 617 in Alzheimer’s disease, 82 apoptosis, 485, 487 aquaporin, 237 arachidonic acid, 307 archaea genome, 109 arginase inhibition by Lys, 429 arginine, 429 pKa, 28 arsenate, 201

arsenite, 243 arteriosclerosis, 338 artery intima, 374, 387 arthritis, 334, 347 rheumatoid, 508 artificial insemination, 564 ascites, 398 ascorbate, 46, 255, 294, 313–314 Ashkenazi Jews, 511 asparaginase, 418 asparagine synthetase, 418 aspartate pKa, 28 transaminase, 209 aspirin, 481 asthma, 276, 476, 617 ataxia, 261, 268 Friedreich’s, 261 ataxia-telangiectasia, 116, 261 atherosclerosis, 374, 387, 617 Atkins-diet, 415, 583 ATP, 165–167 citrate lyase, 368 synthase, 252 atresia, 264 atrial natriuretic factor, 277 attention deficit disorder, 322, 611 autism, 611 autocrine, 271 autosomal, 233 autosomal macular dystrophy, 291 autosome, 109 autosplenectomy, 226 avidin, 316 azaserine, 468 azide, 252 bacteria genome, 109 bacteriophage

651

Index

lambda, 103 T4, 102 baldness, 266 barbiturate, 542 BARK, 277 baroreceptor, 393 Barr-body, 512 Barr body, 123 basal metabolism, 382, 405, 448 base, 9–10, 91 basement membrane, 401 Bayes theorem, 240 bean broad or fava, 516 Beckwith-Wiedemann syndrome, 269 benzene, 114 benzoate, 413 Beri-Beri, 285, 300 bicarbonate, 174 biguanides, 481 bile, 287, 289, 293, 298, 302, 311, 334, 337, 343, 363, 370, 397, 459 acid, 370 bilirubin, 255, 397, 459 direct, 461 indirect, 461 biliverdin, 459 biotin, 245, 304, 316–317 biotransformation, 535 birth defect, 611 2,3-bisphosphoglycerate, 223, 581 biuret, 66 blindness color, 265 congenital, 265 blood, 289, 293, 301, 308, 309, 311, 317, 318, 323, 333, 334, 336, 343 brain barrier, 582 coagulation, 213–217 group, 219–221 pressure, 393 blood clotting, 296, 320, 333

652

blood group, 58 blood pressure, 322, 324, 337 Bloom syndrome, 116 blot dot, 192–194 eastern, 183 northern, 183 Southern, 182, 191, 237 western, 183 Bohr-effect, 224 bond acetal-, 12 covalent, 3 disulfide, 47 glycosidic-, 12 hydrogen, 4, 47 hydrophobic, 47 ketal-, 12 peptide-, 34–89 polar, 5 salt, 4, 47 van der Waals, 47 van der Waals, 5 bone, 209, 257, 287, 291, 294, 298, 319, 332, 333, 336 Bordetella pertussis, 275 Bordetella pertussis, 63 bowel, 210, 264 Bowman’s capsule, 400 bradykinin, 277 brain, 210, 299–302, 308, 309, 311, 314, 316, 318, 323, 324, 334, 337, 341, 343, 347, 375, 582 branching enzyme, 351, 519 BRCA, 494 breast feeding, 319 Briggs, G.E., 140, 142 Bruton agammaglobulinemia, 509 Buchner, E., 138 Buchner, H., 138 buffer, 140, 173 burns, 208

Index

Ca-ATPase, 173 cadaverine, 432 caffeine, 274, 476 calciferol, 291–294 calcitonin, 275, 293, 321 calcitriol, 291, 399 calcium, 275, 291, 298, 309, 313, 319–322, 353, 475, 476, 480, 488 channel blocker, 276 calmodulin, 276, 277 cAMP, 101, 274–275, 351, 353, 364, 368, 488 cancer, 112, 206, 208, 209, 254, 267, 269, 287, 296, 313, 317, 318, 344, 347, 469, 490–495 bladder, 494 bone, 209 brain, 494 breast, 493, 494 colon, 493, 494 esophagus, 494 ovarian, 494 pancreas, 494 prostate, 494 throat, 494 virus-induced, 493 capping, 116 capsid, 102 carbohydrate, 11–12, 323, 338 energy content, 447 metabolism, 349–358, 449–450 carbon monoxide, 222, 252, 459 tetrachloride, 398, 462 carbonic anhydrase, 156, 225 carboxylase, 316 carboxylic acid, 5 carboxypeptidase, 442 carcinogen, 112, 490 carcinoma, 490 colorectal, 495 cardiomegalia glycogenica, 519

caries, 479 carnitine, 364 acyl transferase deficiency, 367 deficiency, 367 carnosine, 406 caroten, 392 carotene, 289 carotenodermia, 289 carpal tunnel syndrome, 309 carrier, 234, 238, 512 casein, 476 catabolic, 447 catalase, 162, 255 catalysis, 22 acid/base-, 162 catalytic perfection, 144 catalytic triad, 165 cataract, 265, 355, 517, 612 catecholamine, 425 CD4, 500 CD8, 500 cDNA, 189 chip, 551 CDP, 368 Cech, T., 137 cell adhesion molecule, 488, 500 antigen-presenting, 500 culture, 486 cycle, 485–491 motility, 488 natural killer, 503 phagocytic, 500 shape, 488 cellulose, 12, 445 centromere, 110 ceramide, 369 cerebrohepatorenal syndrome, 534 cerebroside, 582 cerebrospinal fluid, 582 ceruloplasmin, 204, 207, 329, 333, 334 cerumen, 266

653

Index

cGMP, 277 channel voltage-gated, 276 chaperonin, 55 chaperons, 55 Charcot-Marie-Tooth syndrome, 260 checkpoint, 485 G1, 486, 487, 492 cheese, 137 chenodeoxycholic acid, 370 chimera, 548 chip cDNA, 551 DNA, 550 chloramphenicol, 100 chloride, 324 chloride shift, 324 cholecystitis, 397 cholecystokinin, 397 cholera, 275 cholestasis, 397, 462 cholesterol, 306, 307, 313, 325, 333, 334, 370–376, 390–393, 397, 476 ester transfer protein, 374 cholic acid, 370 cholinesterase, 208 choroid plexus, 582 Christmas disease, 216 chromatin, 110 chromatography affinity, 70 ion exchange, 70 reversed phase, 70 size exclusion, 70 chromium, 268 chromosome, 109 abberation, 121–135 acrocentric, 121 banding, 122 breakage, 131 breakage syndrome, 116 deletion, 131

654

inversion, 133 iso-, 132 metacentric, 121 murder, 127 painting, 122 rearrangement, 131–135 ring, 132 submetacentric, 121 telocentric, 121 translocation, 133 X, 121, 123, 512 Y, 121 chronic granulomatous disease, 581 chylomicron, 363, 373, 375, 396, 576 chylomicrons, 289, 293, 296, 451 chymotrypsin, 60, 442 circular dichroism, 74 cirrhosis, 505 citrate synthase, 244 citric acid cycle, see Krebs-cycle cleft palate, 291, 302, 613 clotting, 203, 207 co-dominant, 233 co-lipase, 442 co-substrate, 165 co-transport, 172 cobalamine, 309–311, 317, 320 cocaine, 208, 473, 612 Cockayne syndrome, 115 code genetic, 98 Codex Hamurabi, 137 codon stop-, 29 coenzyme, 165–169 A, 168 Q, 250 coenzyme A, 306 colchicine, 121, 469 Coley’s anemia, 227 colic, 397 collagen, 45, 257, 313, 333, 334

Index

colon, 445 colostrum, 476 coma hepatic, 399 common variable immunodeficiency, 508 complement, 207, 497, 505, 595 complex initiation, 99 condensation, 6 conjugation, 105 constant equilibrium, 19 contact inhibition, 486 contraceptives, 308, 317 cooperativity, 146 copper, 168, 207, 250, 255, 333–336, 338, 343, 473 Cori-Forbe-disease, 519 cornea, 356 coronary heart disease, 387–393 corpus luteum, 474 corticosteroid, 130, 205 Counseling genetic, 560 CRABP, 289 craniosynostosis, 235 CRBP, 289 Cre/LoxP, 549 creatine, 432 kinase, 210, 259, 432 creatinine, 399, 432 CREB, 274 Cri-du-cat syndrome, 131 Crigler-Najjar syndrome, 462 Crohns disease, 336 crossing-over, 105, 111 croton oil, 276 Cruzons craniosynostosis, 258 cryoprecipitate, 208 crystallins, 55 CTAB, 70 CTP, 167

Cutis laxa, 334 cyanide, 252 cyanosis, 225 cyclin, 60, 485–487 A, 492 D, 490, 492 dependent protein kinase, 487, 492 cycloheximide, 100 cysteine, 47 pKa, 28 cystic fibrosis, 193, 264, 296, 558, 560, 565 cystinuria, 435 cytidine triphosphate, see CTP cytochrome, 250 c oxidase, 251 oxidase, 254 P-450, 457 cytochrome c oxidase, 333, 336 cytochrome P450, 537 cytokine, 490, 595 cytomegalovirus, 612 deacetylase, 589 deaf-mutism, 265 deafness, 612 congenital, 265 deaminase, 307 debranching enzyme, 352, 519 decarboxylase, 307 dehydration, 207 dehydrogenase, 299, 303 deletion, 113 deoxycholic acid, 370 deoxyribonucleotides, 467 dermatomyositis, 259 desoxynojirimycin, 57 detergent, 55, 72 deuteranopia, 265 Dewar, Sir James, 16 dextran, 479 diabetes, 57, 337, 401, 518, 595–604, 611, 612, 616

655

Index

bronze, 267, 332 gestational, 384, 387 insipidus, 237 mellitus, 374, 376, 383–388, 401, 508 MODY, 597 type 2, 277, 382 diacylglycerol, 275, 368 dialysis, 68 diarrhea, 291, 296, 307, 323–325, 334, 336, 338, 343, 347, 476, 517 diastereomer, 8, 11 Dicer, 550 dicoumarol, 216 diet protein-saving, 398 diffusion, 170 facilitated, 171 difluoromethylornithine, 435 DiGeorge syndrome, 509 DiGeorge syndrome, 614 digestion, 439–447 digoxin, 394 dihydrobiopterine reductase, 425 dihydrofolate reductase, 421 dihydrotestosterone, 130, 513 dimethylallyl-pyrophosphate, 370 2,4-dinitrophenol, 253 diphtheria toxin, 117 dipole, 4 disaccharide, 12 disease genetic, 112 sex-influenced, 513 sex-limited, 513 X-linked, 512 Disse space of, 373 disulfiram, 453 diuretic, 156, 323–325 DNA, 91–105 amplification, 183

656

chip, 550 cloning, 187–189 complementary, 189 damage, 487 depurination, 115 diagnostics, 190 fingerprinting, 186 junk, 110 ligase, 115, 181 methylation, 115 mobile, 111 polymerase, 115, 183, 485, 486 probe, 181 repair, 114–116 nucleotide excision, 114 repetitive, 110 replication, 485 satelite, 110 sequencing, 185 structure, 91–94 DNase, 102 Domagk, G.J.P., 421 domain, 47 dominant, 233 dominant negative, 512 Down syndrome, 124 Down syndrome, 124, 135, 560, 617 Down’s syndrome, 336 Dubin-Johnson syndrome, 463 Dumas, 285 dwarfism, 235, 257, 258 dysbetalipoproteinemia, 376 dysosteogenesis, 258 dysostosis, 258 dysplasia skeletal, 258 dystrophy muscular, 169 EC code, 138 Escherichia coli, 275 eczema, 476

Index

edema, 203, 205, 208, 300, 398, 400, 401 Edman-sequencing, 74 Edward syndrome, 124 Edward syndrome, 126 Eflornithine® , 435 egg, 289, 298, 301, 308, 316, 336 Ehlers-Danlos-syndrome, 45, 257 elastase, 442 elastin, 307, 333, 334 electrolyte, 323, 399 electronegativity, 4 electrophoresis, 70, 204, 206, 208, 209, 226 2D, 553 immuno-, 205 electroporation, 547 element chemical, 3 ELISA, 501 elliptocytosis, 263 elongation factor, 117 emphysema, 206 enantimer, 8 end product inhibition, 146 endocrine, 271 endocytosis, 206, 277 of virus, 103 endonuclease restriction, 181 endopeptidase, 442 endoplasmic reticulum, 535 energy, 15 activation, 19, 22 free, 18, 249 of reaction, 15 requirement, 448 Engelhart, J.F., 137 enhancer, 117, 131 enrichment, 285 entero-hepatic circulation, 370 enteropathy, 207 enteropeptidase, 447 enthalpy, 17

envelope, 102, 103 enzyme, 137–179 class, 138 commission, 139 diagnostic, 208 inactivation, 157–159 inhibition, 150–157 competitive, 150 mixed, 157 noncompetitive, 154 uncompetitive, 154 kinetics, 140–150 naming of, 138 epidermolysis bullosa, 46, 264 epigenetics, 589 epilepsy, 156, 611 epimerase, 355 epinephrine, 275, 277, 353, 364, 402, 570 equilibrium, 15 ER, 275, 370 erb B, 492 erythroblastosis fetalis, 220 erythrocyte, 57, 201, 209, 294, 296, 308, 318, 323, 324, 342, 343, 347, 356, 375, 517, 581 erythromycin, 100 erythropoietin, 189, 399, 489, 490 ester, 11 carboxylic, 6 phosphate, 6, 10 thio-, 7 ethanol, 300, 301, 304, 308, 311, 317, 325, 336 forensic determination, 143 in protein precipitation, 68 ether, 7 ethidium bromide, 114 ethylene oxide, 114 euchromatin, 110 eukaryota genome, 109 evolution

657

Index

convergent, 518 exercise, 321 exocytosis, 276 exoglycosidase, 369 exon, 109 exonuclease, 94 exopeptidase, 442 expressivity, 234 extein, 60 eye, 287, 289, 302, 334, 337 Fab -fragment, 497 factor elongation, 99 initiation, 99 factor VIII, 189 FAD, 47, 167, 301 Fanconi anemia, 116 Fanconi-Bickel-disease, 520 fat, 11, 364 energy content, 447 metabolism, 450 fatty acid, 11, 311, 316 biosynthesis, 367–368 branched-chain, 366 essential, 367 mono-unsaturated, 11 odd-chain, 366 poly-unsaturated, 11 saturated, 11, 361 trans-unsaturated, 389 unsaturated, 361, 476 favism, 357 Fc fragment, 497 feces, 293, 306 feedback inhibition, 455 feedforward stimulation, 455 Fenton-reaction, 314 ferment, 137 ferritin, 255 ferrochelatase, 459 ferroxidase, 333

658

fetal alcohol syndrome, 473 fetoprotein, 207 fetuin A, 322 fever, 402 fibrillin, 258 fibrin, 213, 214 fibrinogen, 203, 204 fibrocystic breast disease, 294 fibronectin, 501 ficin, 137 Fischer, E., 140 FISH, 122, 131, 186 fish, 293, 300, 304, 306, 309, 320, 333, 336, 338, 345 flatulence, 445 flavin adenine dinucleotide, see FAD flavin mononucleotide, see FMN flavonoid, 393 fluoride, 201 fluorine, 332–333, 342 fluoroacetate, 245 fluorouracil, 468 Flynn effect, 479 FMN, 167, 301 foam cell, 374 folate, 313, 317–318, 336, 421, 422, 468, 473, 474, 481, 614 folliculosis, 291 fortification, 285 fos, 492 founder effect, 556 fragile X syndrome, 261 frequency allele, 555 gene, 555 fructoaldolase, 518 fructokinase, 354, 518 deficiency, 354 fructose, 449, 518 intolerance, 354 metabolism, 354 fructose-1,6-bisphosphatase, 349

Index

deficiency, 355 fructose-1,6-bisphosphate, 351 fructose-2,6-bisphosphate, 351 fructosuria, 354, 518 fruit, 298, 308, 314, 317, 323 Funk, 285 G-protein, 272, 273, 490 galactitol, 355, 517 galactocerebroside, 582 galactonate, 517 galactosaemia, 355 galactose, 11, 100, 449 metabolism, 355 galactosemia, 517 galactosidase, 100 gall stone, 371, 382, 397 ganglioside, 369, 582 gastroenteritis, 477 Gaucher’s disease, 369 Gaussian distribution, 608 gel filtration, 70 gene, 91–105, 109, 111–112 candidate, 187, 616 duplicated, 111 mapping, 185–187 pseudo-, 111 regulation, 100 SRY, 123 gene manipulation germline, 549 gene therapy somatic, 551 genetic drift, 556 genome archaea, 109 bacteria, 109 eukaryota, 109 human, 109 organelle, 109 virus, 109 genotype, 233

thrifty, 589 geophagy, 337 Giardia, 337 Gibbs-Helmholtz-equation, 18 glaucoma, 265 in diabetes, 595 globulin, 204 glomerulonephritis, 400, 401 glomerulus, 400, 401 glucagon, 200, 275, 350, 353, 570 glucocerebrosidase, 369 glucocorticoid, 364, 402, 570 receptor, 93 glucokinase, 349, 355 gluconeogenesis, 245, 349–351, 356, 402, 450, 452, 454 glucose, 11, 100, 101, 306, 316, 321, 323, 334, 355, 449, 582 glucose transporter 2, 520 glucose-6-phosphatase, 349, 519 deficiency, 353 glucose-6-phosphate dehydrogenase, 356, 368 deficiency, 558 glucuronic acid, 358 GluT2, 520 glutamate dehydrogenase, 410 pKa, 28 glutaminase, 418 glutamine cycle, 435, 579 in the kidney, 435 synthetase, 418 glutathione, 357 peroxidase, 357 reductase, 357 glutathione peroxidase, 344 glyceraldehyde-3-phosphate dehydrogenase, 201 glycerol, 11

659

Index

phosphate, 364 shuttle, 246 glycine, 370, 418 cleavage enzyme, 418 in collagen, 257 glycogen, 12, 351–354, 402, 449 degradation, 352 phosphorylase, 352, 355 deficiency, 353 storage disease, 353 storage diseases, 519 synthase, 519 regulation of, 352 synthesis, 351 glycogenoses, 519 glycolysis, 199–201, 356, 449 anaerobic, 449 glycoprotein, 203, 220 glycosidic bonds, 12 glycosphingolipid, 369 goiter, 341 gonad, 336 gout, 343, 469 grain, 296, 300, 301, 306, 308, 316, 317, 320, 333, 336, 343, 345 GroES/GroEL, 55 groove major, 92 minor, 92 group aldehyde-, 11 alkyl-, 5 amino-, 6, 10, 27, 34 carbonyl-, 6, 12 carboxy-, 5, 9, 10, 27, 34 guanidino-, 28 hemiacetal-, 6, 12 hemiketal-, 12 hydroxy-, 5, 12 imidazole-, 28 keto-, 11 selenol-, 28

660

sulfhydryl-, 6, 10 growth, 405 growth factor, 488 epidermal, 486, 489 fibroblast, 489 insulin-like, 489 nerve, 489 platelet-derived, 486, 489, 492 receptor, 492 transforming, 489 growth hormone, 189, 364 GTP, 99, 167 guanosine triphosphate, see GTP guanylate cyclase, 277 gyrase, 95 Hageman factor, 214 hair, 291, 337 Haldane, J.B.S., 140, 142 haploinsufficiency, 236 haplotype, 237 haptoglobin, 204–206 Hardy-Weinberg equation, 555–556 Hartnup’s disease, 261 Hartnup’s disease, 436 hawkinsinuria, 427 HbA1c , 57 HDL, 451 heart, 209, 210, 267, 300, 324, 325, 333, 334, 347, 393 defect, 614 heat, 15 capacity, 15 of evaporation, 18 Heinz-bodies, 517 helicase, 95 helix alpha-, 39 amphipatic, 39 DNA, 92 helix-loop-helix, 118 helix-turn-helix, 118

Index

HELLP-syndrome, 533 hematocrit, 203 heme, 47, 168, 206, 250 degradation, 459–463 oxygenase, 459 synthesis, 457–459 hemiacetal, 6, see group,hemiacetalhemicellulose, 445 hemiketal, see group,hemiketalhemizygote, 512 hemochromatosis, 207, 267 classic, 267 juvenile, 268 hemoglobin, 137, 146, 206, 221–227, 333, 517 -C, 226 adult, 225 carbamino-, 225 degradation, 459 embryonal, 225 fetal, 221, 225 glucated, 57 H disease, 227 Lepore, 269 sickle cell, 226 hemolysis, 205, 206, 462 hemopexin, 204–206 hemophilia, 263 A, 216, 512 B, 216 hemostasis, 213 Henri, V., 140 heparin, 214, 216, 363 hepatitis, 208, 398, 462, 565 hepatoblastoma, 269 hepatocyte, see liver hepatomegaly, 517 heptose, 11 hereditary fructose intolerance, 518 Hereditary non-polyposis colon cancer, 115 hermaphrodism pseudo-

female, 129 male, 130 true, 129 Hers disease, 520 heterochromatin, 110 heterogeneity allelic, 235, 237 locus, 234 heteroplasmy, 234 heterotropic, 146 heterozygote advantage, 556 compound, 233 hexokinase, 200 hexosamidase A, 369 hexose, 11 Hill-coefficient, 146 hippurate, 413 histamine, 275–277 histidine, 429 pKa, 28 histone, 110, 117 HIV, 193 HMG-CoA, 370 reductase, 373 Homer, 137 homocysteine, 422 homocysteinuria, 422 homogentisate, 427 homotropic, 146 homozygous, 233 hookworm, 337, 611 hormone, 271–277 receptor, 271–273 tyroid, 272 Hsc70, 55, 323 Hsp70, 55 human genome, 109 Hunter disease, 527 Huntington’s disease, 83, 236, 262, 558 Hurler-Scheie disease, 527

661

Index

hybridization, 96 hydrocephalus, 291 hydrogen peroxide, 253, 581 hydrogen sulfide, 252 hydrolysis, 6 hydroperoxide radical, 253 hydrops fetalis, 220 hydroxy radical, 253 hydroxyapatite, 319, 332 hydroxybutyrate, 366 7α-hydroxylase, 370 hydroxymethylcytosine, 102 hyperammonemia, 582 hyperbilirubinemia, 302, 461 conjugated, 462 mixed, 462 unconjugated, 462 hypercholesterolemia, 374, 375 familial, 236, 375 hyperchylomicronemia, 375 hyperglycemia, 582 hyperglycinemia non-ketotic, 418 hyperlipidemia, 401 hyperlipoproteinemia, 375 hyperlysinemia/uria, 429 hyperparathyroidism, 209 hypertension, 382, 393–396, 400, 401 portal, 398 hypertriglyceridemia, 376 hyperuricemia, 469 hypoalbuminemia, 401 hypochlorite, 253, 581 hypochondroplasia, 258 hypoglycemia, 518, 582 hypoketotic, 367 hypophosphatemia, 259 hypothalamus, 341, 477 hypotonia, 517 hypoxia, 253 induced transcription factors, 61 response element, 61

662

I-cell disease, 57, 521 icterus, see jaundice IDL, 451 IEF, 553 IgA, 476 ileum, 370 Iliad, 137 immune system, 291, 307, 316, 318, 333, 337, 338 immunoglobulin, 497–501, 506–507 A, 499 D, 499 E, 294, 499 G, 497, 499 M, 499 subclass deficiency, 508 imprinting, 235, 268 in-vitro fertilization, 565 inactivation X-, 512 inbreeding, 511, 557 induced fit hypothesis, 163 infant, 319 infarct, 169, 208, 209, 226 myocardial, 387 infertility, 123 inflammation, 206 INK4a, 492 inositol-1,4,5-trisphosphate, 275 insertion, 113 insulin, 189, 200, 268, 276, 320, 336, 350, 363, 364, 368, 383, 569 resistance, 277 insulin receptor substrate, 597 integrase, 103, 104 integrin, 488 intein, 60 interactome, 145, 591 interferon, 189, 490, 505, 595 interleukin, 294, 490 intermittent claudication, 294

Index

intestine, 209, 287, 289, 293, 296, 298, 300, 301, 304, 308, 309, 314, 317, 321, 323, 325, 334, 336, 337, 341, 343, 442 brush border, 443 intrinsic factor, 309, 311 intron, 109–111 iodine, 338–342 ion channel ligand gated, 272 IQ, 479, 609 iron, 168, 250, 255, 311, 313, 325–333, 338, 473, 475, 480 deficiency, 207 poisoning, see hemochromatosis iron-sulfur protein, 168, 250 ischemia, 201, 253, 449 isocitrate dehydrogenase, 244 isoelectric focussing, 72 isoelectric point, 30 isoenzyme, 37, 169, 208 isomer, 8, 11 cis-trans, 8 geometric, 8 optical, 8 positional, 8 isoniazid, 303, 304, 308 isopentenyl-pyrophosphate, 370 isoprene, 286 isoprenoid, 370 janus kinase, 490 jaundice, 398, 461, 517 obstructive, 208, 209 jun, 492 karyotype, 121–123, 131 Kashin-Beck-disease, 347 katal, 143 kernicterus, 220, 461 Keshan disease, 347 ketoglutarate dehydrogenase, 244

ketone, 12 ketone bodies, 366, 577 kidney, 207, 209, 226, 237, 289, 291, 293, 301, 302, 314, 316, 317, 321, 324, 325, 333, 341, 342, 393, 399–402 disease polycystic, 264, 401 stone, 471 kinetics, 15 reaction, 19–21 Klinefelter syndrome, 127 knock down, 550 in, 549 out, 548 conditional, 549 Koshland, D.E., 163 Krabbe’s disease, 370 Krebs-cycle, 199 Krebs-cycle, 244–247, 449 Kuppfer cell, 397 kwashiorkor, 577 β-lactamase, 105 lactate, 325 lactate dehydrogenase, 201, 209 lactation, 293, 314, 338, 356, 405, 476–479 lactoferrin, 476 lactoglobulin, 476 lactose, 12, 100, 321, 476 intolerance, 518 intollerance, 445 permease, 100 lamin, 485 Lavoisier, 285 LCHAD, 533 LDL, 451 Le Chatelier’s principle, 21 lead poisoning, 459 leavening (of bread), 337 Leber hereditary optic neuropathy, 531 lecithin-cholesterol acyl transferase, 374

663

Index

legume, 300, 304, 316, 333, 336, 343 Leigh-syndrome, 528, 531 leptin, 490, 595 Lesch-Nyhan syndrome, 469 leucine zipper, 118 leucocytes, 477 leukemia, 125, 494 lymphoblastic, 125 myelogenous, 125 Lewis acid, 168 Lewy-bodies, 83 Li-Fraumeni syndrome, 494 library genomic, 188 Limbic, 285 light, 291 polarized, 9 lignin, 445 likelihood ratio, 187 Lind, 314 LINE-1, 110–112 Lineweaver-Burk-transformation, 143 linkage, 186 linoleic acid, 307, 361, 476 linolenic acid, 361 lipase, 210, 320, 442 adipose tissue, 364 hepatic, 373 pancreatic, 363 lipid, 320 metabolism, 361–376 lipid storage disease, 369 lipofuscin, 296 lipoic acid, 243 lipoprotein, 363, 364, 371–376, 451 HDL, 373–375 high density, 338 IDL, 373 LDL, 373–375 lipase, 363, 373 low density, 296

664

VLDL, 364, 368, 373, 375 liposome, 73, 552 Lisch nodules, 495 lithium, 612 lithocholic acid, 370 liver, 169, 203, 206, 209, 215, 267, 289, 291, 293, 296, 298, 301, 309, 311, 313, 314, 316, 317, 334, 338, 342–344, 352, 355, 370, 396–399, 402, 452, 577 cancer, 207 cirrhosis, 207, 208, 398 infantile, 206 enzymes, 415 fatty, 398 locus, 233 heterogeneity, 234 lod score, 187 lordosis, 258 low birth weight, 473, 612 Lowry, 66 lung, 206, 264, 294 Lunin, 285 lymph, 289, 363 lymphocyte, 207 B, 507 T, 507 lymphocytes T-, 294 Lyons hypothesis, 123 Lyons-hypothesis, 512 lysine, 429 pKa, 28 lysis, 103 lysogenic, 103 lysosome, 277, 354, 369, 506, 519 lysozyme, 102, 439, 477 lysyl oxidase, 333, 334 macroglobulin, 206 macrophage, 373, 374, 477 macula degeneration, 265

287, 308, 333, 356,

Index

magnesium, 99, 324–325, 473 Maillard-reaction, 59 malaria, 51, 226, 558, 611 G6PDH deficiency in, 516 malate aspartate shuttle, 246 malic enzyme, 368 malnutrition, 207 malonyl-CoA, 367 maltose, 12 mammary gland, 356 manganese, 168 manic-depressive, 611 manifestation, 512 mannose, 11 maple syrup urine disease, 422 Marfan syndrome, 257 marker linked, 237 Maroteaux-Lamy disease, 527 mass molecular, 3 mass action law of, 19 mass element, 319–325 mass spectrometry, 74, 553 matrix protease, 60 Maxam-Gilbert method, 185 McArdle disease, 520 McArdle’s disease, 353 Mdm2, 493 meat, 298, 300, 304, 306, 308, 309, 320, 323, 336, 343, 345 megadose, 285 meiosis, 105, 111, 121, 124 MEK, 490 melanin, 334, 336 membrane protein, 39 Mendel, Gregor, 233 Menke’s disease, 334 Mennonites, 511 mental retardation

fragile X, 261 Menten, M.L., 140 mercury, 612 MERRF-syndrome, 532 metabolic syndrome, 594 metabolome, 591 metachromatic leukodystrophy, 370 metallothionein, 255, 334, 336 metaphase, 121 metastable, 22, 53 metastasis, 491 methanol, 151 methemoglobin, 221, 225 reductase, 221, 581 methionine, 422 methotrexate, 151, 421, 468 methyl bromide, 114 methylation, 61, 117 RNA-, 97 methylene blue, 222 methylmalonic aciduria, 425 methylmalonyl-CoA mutase, 310 mevalonate, 370 MHC, 501 class I region, 503 class II region, 504 class III region, 505 polymorphism, 508 micelle, 371 Michaelis, L., 140 microarray, 550 microcephaly, 612 microsatellite, 110 milk, 289, 293, 298, 300, 301, 309, 314, 319, 333 allergy, 476 composition, 476 millet, 304 mineral, 318–347 mineralocorticoid, 130 minisatellite, 110

665

Index

mitochondria, 41, 60, 109, 199, 234, 243, 364, 366, 449 inherited diseases, 528–533 mitogen, 486, 488–491 mitosis, 121, 131, 485 S-phase, 114 mitral valve, 257 molten globule, 53 molybdenum, 342–344 monosaccharide, 11 monosomy, 124 Montezuma’s revenge, 275 Morgan, T.H., 186 Morquio disease A, 527 B, 527 mosaicism, 512 motive, 49 mountain sickness, 156 mRNA processing, 116 mucous membranes, 287 Muenke’s craniosynostosis, 258 Mulder,G.J., 137 muscle, 201, 209, 210, 276, 294, 296, 300, 314, 320, 323, 324, 333, 352, 375, 402, 449, 578 muscular dystrophy, 208, 259, 260, 296 Becker, 259 Duchenne, 259, 557 Duchenne, 192 limp-girdle, 260 myotonic, 260 mutagen, 112, 254, 490 mutagenesis insertional, 547 mutation, 109, 112 dominant, 236 dominant negative, 236 frameshift, 113 gain of function, 236 haploinsufficient, 236

666

loss of function, 236 missense, 113 nonsense, 113 point, 113 rate, 557 recessive, 236 silent, 113 site directed, 190 myasthenia gravis, 259 myc, 492, 493 myelin, 582 myeloma multiple, 509 myoglobin, 221 myokinase, 167 myopathy, 336 ragged red fibre, 532 myositis, 208, 259 myxedema, 341 Na+ /K+ -ATPase, 322 Na/K-ATPase, 172 NAD, 167, 303, 304 NADH oxidase, 254, 581 Q reductase, 251 NADP, 303, 304 necrosis, 206, 208 neoplasia, 490 nephritis glomerular, 401 nephron, 400 nephrotic syndrome, 207, 401 neu, 492 neural tube, 474, 612, 614 neurofibromatosis, 495 neuropathy, 260 neuropeptide Y, 587 neurotransmitter, 271 niacin equivalent, 304 nicotinamide, 169, 304–307, 316 production from Trp, 427

Index

Nicotinamide-adenine dinucleotide, see NAD nicotine, 303 Niemann-Pick disease, 370 ninhydrin, 67 nitric oxide, 277 synthase, 277 nitroglycerin, 277 nitrous acid, 114 nojirimycin, 57 norepinephrine, 364, 402, 570 normal distribution, 608 nuclear magnetic resonance, 74 nuclease, 97, 443 nucleoside, 91 nucleosome, 110 nucleotide, 91 nut, 300, 304, 316, 333 nutrient pool, 283 oasthouse disease, 436 obesititis, 595 obesity, 376, 381–384, 394, 397, 479 occur, 287, 291, 296 ochronosis, 427 Okazaki-fragment, 95 oligo allele-specific, 550 oligomycin, 253 oligosaccharide, 12 oncogene, 491 cellular, 491 proto-, 491, 493 viral, 493 oncotic pressure, 203 oogonia, 124 operator, 101 operon lac-, 100–101 Trp-, 101 order of a reaction, 19 organelle genome, 109

organophosphate, 208 Ori-c, 95 oriental flush, 453 ornithine decarboxylase, 435 orotic aciduria, 464 osteitis deformans, 209 osteoarthritis, 382 osteoblast, 209, 287 osteocalcin, 298 osteoclast, 287 osteogenesis imperfecta, 45, 237, 257 osteomalacia, 209, 294, 322 osteoporosis, 322, 332, 334, 617 ouabain, 394 ovary, 517 oxalate, 321 oxaloacetate, 454 oxanion hole, 165 oxidative phosphorylation, 249–253, 449 oxidoreductase, 301 oxygen, 250, 285 reactive, 253–255 toxicity, 254 oxytocin, 477 p53, 487, 492, 493, 553 Paget’s disease, 208 palindrome, 97, 181 pancreas, 169, 264, 276, 287, 289, 298, 311, 316, 337, 442 pancreatitis, 208, 210, 447 pantothenate, 306–307 pantothenic acid, 169 papain, 497 paracrine, 271 paraprotein, 205 paraproteinemia, 509 parathormone, 291, 293, 321 parathyroid, 325 Parkinson’s disease, 83 Patau syndrome, 124

667

Index

Patau syndrome, 126 PBX2, 505 PCR, 183–184, 193 pedigree, 238 pellagra, 436 penetrance, 234, 238, 512 penicillin, 105 penicillinase, 105 pentachlorophenol, 253 pentose, 11 phosphate pathway, 355 PEP-carboxykinase, 349 pepsin, 442, 447 peptide, 34 signal, 60 permanent wave, 41 pernicious anemia, 311, 318 peroxidase, 255 peroxisome, 366 peroxisomes inherited diseases, 533–535 pesticide, 83 petechia, 314 pH, 9 in protein precipitation, 68 phage, 188 phagocytosis, 497 phenobarbital, 459, 462 phenocopy, 235, 511 phenol, 10 phenome, 591 phenotype, 233 phenylacetate, 413 phenylalanine, 425 hydroxylase, 425 phenylketonuria, 266, 612 phenytoin, 612 phocomelia, 612 phorbol ester, 276 phosphatase I, 353 phosphate, 173, 259

668

phosphatidic acid, 368 phosphatidyl inosititol, 275 inositol, 213 serine, 213 phosphoanhydride, 165 phosphodiesterase, 274, 277 phosphofructokinase, 146, 200, 349, 351 M isoform, 520 phosphoglyceride, 368 phospholipase, 102, 368, 443 C, 275, 488, 489 phospholipid, 333 phosphorus, 291, 319–322 phosphorylase, 520 phosphorylation, 61, 117, 351, 364, 448, 485, 487, 489, 569 phototherapy, 302, 462 phylloquinone, 296 phytanic acid, 367 phytate, 321, 325, 336, 337 phytohemagglutinin, 121, 486 pI, 70 pica, 306 pinocytosis, 203 pituitary, 341 placenta, 209, 474, 497 plaque, 374 atheromatous, 387 senile, 617 plasma, 203, 207 plasma cell, 203 dyscrasia, 509 plasmid, 104, 188 plasmin, 214 plasminogen, 214 plasminogen activator inhibitor 1, 595 platelet, 207, 213, 216 pleiotropy, 234 polyadenylation, 116 polyamines, 432 polycistronic, 101

Index

polycythemia, 225 polymerase DNA, 94, 112 RNA-, 97 polymorphism, 556 polyol, 358 polyphenol, 589 polyphenolic, 393 polyploidy, 123 polysaccharide, 12 Pompe disease, 519 Pompe’s disease, 354 porphyria, 457 acute intermittent, 236, 457 cutanea tarda, 459 porphyrin, 457 porphyrinogen, 457 potassium, 322–324 Prader-Willi syndrome, 268 pregnancy, 209, 287, 293, 300, 309, 314, 318, 337, 341, 405, 473–476 primaquine, 357 primase, 95 primer, 183, 184 prion, 77–80 proband, 233 probenicid, 471 processing RNA-, 97 proenzyme, 60 progesterone, 376 progestin, 130 prolactin, 477, 490 proline, 429 prolyl hydroxylase, 61 promotor, 101, 117, 131, 276 Prontosil, 421 proofreading, 94 prophage, 103 prophase, 111, 122 propositus, 233 prostaglandin, 271, 316

prostate cancer, 209 prosthetic group, 165 protanopia, 265 protease inhibitor, 206 serine, 164, 206, 214 proteasome, 504 protein, 34–89, 137, 307, 323 -denaturation, 55 acetylation, 61 as buffer, 174 band 3, 263 Bence-Jones, 509 biological value of, 406 C-reactive, 206 concentration, 66 constitutive, 100 energy content, 447 heat shock, 506 hydroxylation, 60 inducible, 100 intrinsically disordered, 50, 74 kinase, 61, 274 A, 276 C, 276 G, 277 mitogen activated, 490 membrane purification, 72 metabolism, 452 methylation, 61 phosphatase, 61 phosphorylation, 61 RDA, 405 splicing, 60 synthesis, 98–100 tyrosine kinase, 489 proteinuria, 400, 401 proteolysis, 60 proteome, 591 proteomics, 72, 553

669

Index

prothrombin time, 215 protomer, 51 provitamin, 286 pseudocholinesterase, 208 Pseudomonas aeruginosa, 264 pseudouridine, 97 psoriasis, 208, 469 purine biosynthesis, 463 degradation, 464 puromycin, 100 putrescine, 432 pyelonephritis, 401 pyloric stenosis, 613 pyridoxalphosphate, 410 pyridoxin, 307–309 pyrimidin metabolism, 464 pyrophosphatase, 166, 418 pyrrolysine, 29 pyruvate, 199, 454 carboxylase, 245, 261, 349 dehydrogenase, 243, 244, 261 kinase, 200, 349 inherited defect, 201 QH2 - Cytochrome c reductase, 251 quantitative trait loci, 615 R-factor, 105 radiation, 612 ionizing, 113 radical, 253, 294 Raf, 490, 492 RAGE, 505 ragged red fibre, 532 Ras, 490, 492, 495 rate constant, 20 RDA, 281, 283 in pregnancy, 474 reaction

670

0th-order, 20 1st order, 21 2nd order, 21 order, 19 velocity, 19 receptor 7-transmembrane segment, 273 acetylcholine, 272 muscarinic, 277 desensitisation, 277 down-regulation, 277 G-protein linked, 272 GABA-A, 272 glutamate NMDA, 276 muscarinic, 276 phosphorylation, 277 scavenger, 373, 374 tyrosine kinase, 272 recessive, 233, 511 recombination, 105 homologous, 105, 111 redox potential, 249 redox-reaction, 167–168 reductionism, 138 Refsum’s disease, 367 regression toward mean, 609 renal disease end-stage, 401 renin, 393, 399 replicase RNA, 104 replication, 94–95, 112 -fork, 94 resistance antibiotic-, 104 respiratory burst, 581 response element, 118 restriction enzyme, 181 reticuloendothelial, 206 retinal, 287–291 retinitis pigmentosa, 265, 291

Index

retinoblastoma, 494 protein, 486–487, 492, 493 retinoic acid, 272, 287, 288, 612 retinoid, 336, 338 retinol, 255, 294 binding protein, 205 retinol equivalent, 287 retinopathy diabetic, 265 retrolental fibroplasia, 296 retroposon, 111, 112 viral, 112 retrovirus, 493, 552 Rett-syndrome, 513 reverse transcriptase, 111 RFLP, 186, 237 rhabdomyoma, 266 rhabdomyosarcoma, 269 rhesus factor, 220–221 incompatibility, 462 rhizomelia, 258 rhodopsin, 265, 277, 287, 288 RhoGAM, 221 riboflavin, 169, 301–302, 304 ribose-5-phosphate, 355 ribosome, 96, 99 ribozyme, 137 rickets, 209, 259, 294 rifampicin, 99 rigor, 322 RISC, 550 RNA, 96–97 editing, 119 messenger, 96 polymerase, 116, 118 ribosomal, 96, 99 transfer, 96, 98 RNAi, 550 Robertsonian translocation, 134 rotenone, 252 rubella, 612

S-adenosylmethionine, see SAM Sørensen, P.L., 140 saccharose, 518 saliva, 338, 341 salivary glands, 439 salt, 323 in protein precipitation, 68 SAM, 169 Sanfilippo disease A, 527 B, 527 C, 527 D, 527 Sanger dideoxy method, 185 sarcoma, 490, 494 Kaposi, 493 Schiff-base, 59 Schilling-disease, 311 schizophrenia, 306, 611 scleroderma, 505 scurvy, 314 SDS, 70 secretor, 220 seed, 298 seizures, 156 selection, 558 selenium, 255, 294, 344–347 selenocysteine, 29, 255 pKa, 28 senescence, 486 serotonin, 275, 425 serum, 169 sex hormone binding globulin, 205 SH3-domain, 46 sheet,beta-, 41 Shine-Delgarno sequence, 99 shock, 208 sialidase, 506 silencer, 117 silk, 41 sirtuin, 61, 589

671

Index

skin, 291, 293, 296, 306, 308, 314, 316, 337 sleeping sickness, 435 Sly disease, 527 small for date birth, 318 smoking, 374, 388, 394, 476, 612 SNP, 616 snurp, 116 sodium, 322–324, 394 soil, 319 solubilization, 72 sorbitol, 518 sorghum, 304 spectrin, 263 spectroscopy infrared, 74 spermatogonia, 124 spermidine, 432 spermine, 432 spherocytosis, 263 sphingolipid, 220, 369 sphingolipidosis, 369 sphingosine, 369 spina bifida, 337, 474, 614 spleen, 216, 459 spliceosomes, 116 splicing, 116 alternative, 119 protein, 60 spongiform encephalopathy, 77 sprue, 317, 336 src, 492 Staphylococcus aureus, 264 starch, 12 Stargardt-disease, 291 starvation, 576 steatorrhea, 264 stem cells embryonic, 548 steroid, 272, 569 stomach, 304, 309, 311, 333, 334, 338, 343, 442 Strecker-degradation, 59

672

Streptococcus mutans, 479 streptomycin, 100 stringency, 182 structure primary, 74 quaternary, 51, 74 secondary, 37, 74 tertiary, 47, 74 substitution, 113 subunit, 51 succinate dehydrogenase, 244 succinyl-CoA, 418 succinylcholine, 209 sucrose, 12, 479 suicide substrate, 159 sulfanilamide, 421 sulfatase, 369 sulfonamide, 469 sulphite oxidase, 342 sulphonamide, 151, 298, 300, 421 Sumner, J.B., 137 SUMO, 63 supercoiling, 93 superfemale, 128 superoxide, 253, 314 superoxide dismutase, 254, 333 superoxide radical, 581 superoxide themselves, 336 surgery, 402 symport, 172 syndactyly, 258 syndrome metabolic, 594 synuclein, 83 syphilis, 565, 612 system, 16 systemic lupus erythematosus, 401, 505, 612 T-cell receptor, 500 tandem repeat, 110

Index

Tangier disease, 375 TAP1/2, 504 tapeworm, 311 Taq, 183 Tarui disease, 520 taurine, 370, 476 tautomer, 113 Tay-Sachs disease, 369 Tay-Sachs disease, 565 telomerase, 112 telomere, 110, 112 temperature, 15 teratogen, 611 teratogenesis, 291, 294, 302, 318, 337, 343 tetany, 322, 325 tetracycline, 100, 481 tetrose, 11 TGF β, 595 thalassemia, 192, 227, 558, 565 major, 227 minor, 227 thalidomide, 612 thanatophoric dysplasia, 236, 258 theophylline, 274, 276 thermodynamics, 15–19 first law of, 16 second law of, 18 third law of, 18 thermogenesis, 382, 448 Thermus aquaticus, 183 thiamin, 299–301, 304 thiamine, 243, 356 thiazide, 481 thioglycolic acid, 41 thiol, 6 thoracic duct, 363 threonine, 418 thrombin, 214 time, 215 thrombomodulin, 214 thrombosis, 213, 401

thymidylate kinase, 485 thymidylate synthase, 468 thyroid, 293, 301, 311, 325, 338, 341, 344 hormone, 364, 569 thyroxin, 321, 338 thyroxine, 205, 402 tissue factor, 214 plasminogen activator, 189, 214 TNF α, 595 tobacco, 314, 531 tobacco-alcohol amblyopia, 531 tocopherol, 294–296 equivalent, 294 tooth, 291, 294, 314, 319, 332, 342 tophi, 469 topoisomerase, 94, 95 toxin, 104 toxoplasmosis, 612 trace element, 319, 325–338 training, 405 tranquilizer, 293 transaldolase, 356 transaminase, 307 transamination, 408 transcobalamin, 309, 311 transcortin, 204, 205 transcriptase reverse, 104 transcription, 96–97, 116 factor, 118 transcriptome, 591 transcuprin, 334 transducin, 277 transduction, 105 transferrin, 205, 207, 336, 476 transformation, 105 transfusion, 219 exchange, 462 transgenic mice, 547 transglutaminase

673

Index

tissue, 83 transition, 113 state, 22 transketolase, 299, 356 translation, 98–100, 117 translocation, 113 Robertsonian, 124 transport membrane-, 170–173 active, 172 passive, 170 transposon, 111 transthyretrin, 204, 205 transversion, 113 Traube, M., 137 trauma, 206, 402 tri-functional protein, 533 trichloroethylene, 83 triglyceride, see fat Trimethoprim, 421 triose, 11 trisomy, 124 -13, 124, 126 -18, 124, 126 -21, 124 troponin, 275, 276 Trypanosoma brucei, 435 trypsin, 60, 442, 447 tryptophan, 427 tuber, 298, 308 tuberculosis, 209, 611 tuberous sclerosis, 266 tumor progression, 491 supressor, 491 tumor necrosis factor, 506 tungsten, 342–344 Turner syndrome, 128 Turner syndrome, 128, 131, 132 tyrosinase, 263 tyrosine, 425, 427 pKa, 28

674

tyrosinemia I, 427 II, 427 III, 427 tyroxine binding globulin, 205 ubiquinone, see coenzyme Q ubiquitin, 63 UDP-glucuronyl transferase, 462 ultratrace element, 319, 338–347 uncoupler, 253 uniport, 171 urea, 399, 452 cycle, 261 in protein denaturation, 56 urease, 137 uremia, 401, 582 uric acid, 255 uridilylation, 63 uridine triphosphate, see UTP urine, 298, 300, 302, 304, 306, 308, 309, 311, 314, 316, 337, 343, 345 blood in, 206 urobilin, 459 urobilinogen, 459 uroporphyrinogen decarboxylase, 459 uroporphyrinogen I synthase, 457 US-RDA, 283, 285 uterus, 477 UTP, 167 UV, 113 valinomycin, 253 valproic acid, 612 vampire, 306 vas deferens, 264 vasopressin, 237, 275, 276, 402 vector retroviral, 552 vegetable, 289, 298, 300, 301, 314, 316, 317, 323, 338

Index

velocity, 19 Vibrio cholerae, 63, 275 Vibrio parahaemolyticus, 63 Vicia faba, 516 virion, 102, 103 virus, 102–104, 114, 181, 493 adeno-, 552 adenoassociated, 552 animal, 103 genome, 109 papilloma, 493 retro-, 104, 493, 552 RNA, 104 Rous sarcoma, 493 visfatin, 595 vitalism, 138 vitamin, 169, 285–318, 475 A, 362, 392, 473 B3 , 427 B6 , 410, 422, 474 B12 , 422, 442, 481 C, 46, 362, 392, 474, 480, 481 D, 272, 481 E, 255, 362, 375 fat soluble, 286–299 K, 216 M, 421 water soluble, 299–318 vitamin K, 296–299 VLDL, 451, 452 VNTR, 186, 237 von Gierke’s disease, 353 von Gierke-disease, 519 von Recklinghausen’s disease, 495 von Willebrand factor, 216 von Willebrand factor, 213 VopS, 63

intoxication, 323 Watson-Crick structure, 92 weaning, 478–479 weight, 338 whooping cough, 275 Williams syndrome, 132 Wilm’s tumor, 269 Wilson’s disease, 334 Wilson’s disease, 207 Wolff-Chaikoff-effect, 342 work, 15 X-inactivation, 512 X-ray crystallography, 74 xanthin oxidase, 342 xanthine oxidase, 471 xantoma, 375 xenobiotics, 535, 537 xeroderma pigmentosum, 115 YAC, 189 yeast, 293, 316, 317 yellow fever, 398, 462 Zellweger syndrome, 534 Zellweger-syndrome, 366 zinc, 168, 255, 289, 313, 318, 334, 336–338, 442, 453, 473, 475, 476 finger, 93 Zn finger, 118 zymogen, 276, 447

Waldenstroms macroglobulinemia, 509 warfarin, 216, 612 water, 323, 332 anomaly of, 4

675