ENZYMATIC REACTION MECHANISMS Perry A. Frey and Adrian D. Hegeman Universitats- und Lan.j'esbibHothek Darmstadt Bibfiothek Biologio «••*• OXPORD UNIVERSITY PRESS Contents Chapter 1. Enzymes and Catalytic Mechanisms, 1 Catalysis and the Active Site, 1 Rate Enhancement in Enzymatic Catalysis, 3 Conformational Mobility in Catalysis, 5 Substrate-Induced Conformational Changes, 5 Catalysis of Multistep Reactions, 6 Structural Mobility in Enzymes, 6 Acid-Base Catalysis, 9 Acids and Bases, 9 Acid- and Base-Catalyzed Reactions, 11 Nucleophilic Catalysis, 16 Electrophilic Catalysis, 21 Catalysis of Enolization, 21 Imine Formation by Lysine, 23 Catalysis by Metal Ions, 26 Hydrogen Bonding, 30 Strong and Weak Hydrogen Bonds, 30 Hydrogen Bonding in Catalysis, 32 Binding Energy in Catalysis, 34 Binding and Activation Energy, 34 The Active Site as an Entropy Trap, 36 Dissecting the Binding Effect in Enzymatic Action, 40 Stabilization of the Transition State, 41 Binding the Near Attack Conformation, 46 Destabilization of Ground States, 48 Rate Enhancement through Binding of Remote Groups, 48 Characterization of Active Sites, 53 xii Contents Competitive Inhibitors: Analogs of Substrates, 53 Group-Selective Chemical Modification, 53 Site-Directed Mutagenesis, 57 Affinity Labeling, 59 Why Are Enzymes Large Molecules?, 62 Sizes of Enzymatic Binding Domains, 62Catalytic Antibodies, 63 Chapter 2. Kinetics of Enzymatic Reactions, 69 Steady-State Kinetics, 69 One-Substrate Reactions, 70 Two-Substrate Reactions, 74 Three-Substrate Reactions, 89 Isotope Effects, 91 Classes of Isotope Effects, 91 Measurement of Isotope Effects, 95 Transient-Phase Kinetics, 101 Reaction Characteristics, 101 Transient Methods, 102 pH-Rate Profiles, 111 Profile Interpretation, 111 Measurements of pH-Rate Profiles, 111 Allosteric Regulation, 117 Theory, 118 Binding Equations for Cooperative Systems, 120 Aspartate Transcarbamoylase, 123 Chapter 3. Coenzymes I: Organic Coenzymes, 129 Nicotinamide Coenzymes, 129 Structures and Functions of Nicotinamide Coenzymes, 129 Stereospecificity of Hydride Transfer, 132 NAD+ as a Coenzyme, 134 Thiamine Pyrophosphate, 141 Structure, 141 Reaction Mechanism, 141 oc-Lipoamide, 147 Pyridoxal-5'-Phosphate, 148 Enzymatic Reactions Facilitated by Pyridoxal-5'-Phosphate, 149 Pyridoxal-5'-Phosphate-Stabilized Amino Acid Carbanions, 149 Mechanisms of Pyridoxal-5'-Phosphate-Dependent Reactions, 151 Flavin Coenzymes, 158 Structures of Flavin Coenzymes, 158 Mechanisms of Flavin Catalysis, 159 Biotin, 163 Structure and Role as a Carboxyl Carrier, 163 Contents Chemistry of Biotin and A^-Carboxybiotin, 164 Mechanism of Biotin-Dependent Carboxylation, 164 Phosphopantetheine Coenzymes, 165 Structures of Phosphopantetheine Coenzymes, 165 Mechanism of Phosphopantetheine Action, 165 Folate Compounds, 167 Folate Compounds of One-Carbon Metabolism, 168 Enzymes in Tetrahydrofolate Metabolism, 170 Biological Importance of Folate, 171 Amino Acid-Based Coenzymes, 172 Pyruvoyl Decarboxylases, 172 Methylidene Imidazolinone-Dependent Deaminases, 173 Quinoproteins, 174 Chapter 4. Coenzymes II: Metallic Coenzymes, 189 Vitamin B j 2 Coenzymes, 190 Chemistry of B12 Coenzymes, 190 Adenosylcobalamin-Dependent Enzymes, 193 Methylcobalamin-Dependent Enzymes, 199 Heme Coenzymes, 201 Chemistry of Oxygen and Heme, 201 Heme Enzymes, 204 Oxygen Binding and Electron Transfer, 209 Mononuclear Nonheme Iron, 210 Monooxygenases, 210 Dioxygenases, 217 Oxo-Fe2 Complexes, 217 Structures, 218 Reactions of Di-iron Enzymes, 219 Metallopterin Enzymes, 222 Molybdopterin and Tungstopterin, 222 Iron-Sulfur Centers, 227 Structures, 227 Catalytic Functions, 230 S-Adenosylmethionine and Iron-Sulfur Centers, 234 Catalytic Action of S-Adenosylmethionine and [4Fe-4S] Centers, 234 Stoichiometric Reactions of S-Adenosylmethionine and [4Fe^lS] Centers, 236 Divalent Metal Ions, 237 Electrostatic Activation of Coordinated Water, 237 Electrostatic Activation of Enolization, 238 Copper as a Cofactor, 240 Copper Proteins, 240 xiii xiv Contents Other Copper Enzymes, 241 Nickel Coenzymes, 243 Nickel in Methanogenesis, 243 Other Nickel Coenzymes, 245 Long-Range Electron Transfer, 247 Biological Electron Transfer, 247 Marcus Theory, 248 Chapter 5. Enzyme Inhibition, 253 Two-Substrate Analogs, 254 Inhibition and Binding, 254 PALA and Aspartate Transcarbamylase, 254 Suicide Inactivation, 255 Thymidylate Synthase, 255 P-Hydroxydecanoyl Thioester Dehydratase, 260 y-Aminobutyrate Aminotransferase, 262 Kinetics of Slow-Binding and Tight-Binding Inhibition, 268 Slow Binding, 268 Tight Binding, 269 Slow-Binding Inhibition, 270 Dihydrofolate Reductase, 271 Prostaglandin H Synthase, 274 Tight-Binding Inhibition, 280 HMG-CoA Reductase, 280 Alanine Racemase, 285 5-Enolpyruvoylshikimate-3-Phosphate Synthase, 289 Acetylcholinesterase, 291 Chapter 6. Acyl Group Transfer: Proteases and Esterases, 297 Chemistry of Acyl Transfer, 297 Serine Proteases, 300 Chymotrypsin, 301 Subtilisin, 311 Cysteine Proteases, 314 Papain, 315 Caspases, 317 Aspartic Proteases, 317 Molecular Properties, 318 Mechanism of Action, 320 Metalloproteases, 323 Carboxypeptidase A, 324 Thermolysin, 327 Esterases, 328 Structure and Function, 328 Phospholipase A2, 329 Contents Chapter 7. Isomerization, 333 Aldose and Ketose Isomerases, 333 Chemistry, 333 Phosphoglucose Isomerase, 334 Triosephosphate Isomerase, 335 Xylose Isomerase; 341 Phosphomutases, 341 oc-Phosphoglucomutase, 341 P-Phosphoglucomutase, 343 Phosphoglycerate Mutases, 343 Racemases and Epimerases, 346 Proline Racemase, 346 Glutamate Racemase, 350 Mandelate Racemase, 352 UDP-Galactose 4-Epimerase, 355 Ribulose-5-P 4-Epimerase, 360 UDP-/V-Acetylglucosamine-2-Epimerase, 361 Chorismate Mutase, 364 A5-3-Ketosteroid Isomerase, 366 Radical Isomerizations, 368 Glutamate Mutase, 369 Methylmalonyl CoA Mutase, 371 Lysine 2,3-Aminomutase, 376 Newer Isomerases, 379 UDP-Galactopyranose Mutase, 379 Pseudouridine Synthase, 379 Chapter 8. Decarboxylation and Carboxylation, 387 Chemistry of Decarboxylation and Carboxylation, 387 Decarboxylases, 388 Pyruvate Deearboxylase, 389 Amino Acid Decarboxylases, 394 Acetoacetate Deearboxylase, 403 Mevalonate Pyrophosphate Deearboxylase, 405 Radical-Based Decarboxylases, 407 Orotidine Monophosphate Deearboxylase, 414 Carboxylases, 418 Ribulose-1,5-Bisphosphate Carboxylase, 419 Phosphoenolpyruvate Carboxylase, 425 Vitamin K-Dependent Carboxylase, 426 Chapter 9. Addition and Elimination, 433 a,p-Elimination/Addition Reactions, 433 Cofactor-Independent a,P-Elimination/Addition Reactions, 434 Cofactor-Dependent oc,|3-Elimination/Addition Reactions, 440 xv xvi Contents (3,a-Elimination/Addition Reactions, 456 Methylidene Imidazolone-Dependent Elimination and Addition, 456 Carbonic Anhydrase, 462 Isomerization and Elimination, 465 Catalytic Process, 465 Coenzyme B12-Dependent Elimination," 466 Chapter 10. Phosphotransfer and Nucleotidyltransfer, 476 Chemistry of Phosphoryl Group Transfer, 476 Phosphomonoesters, 476 Phosphodiesters, 483 Phosphotriesters, 483 Five-Member Ring Phosphoesters, 484 Enzymatic Phosphoryl Group Transfer, 487 Single and Double Displacements, 487 Phosphotransferases, 489 Protein Phosphorylation: Protein Kinase A, 502 Phosphomonoesterases, 509 Enzymatic Nucleotidyl Group Transfer, 521 Nucleotidyltransferases, 521 Phosphodiesterases, 539 Chapter 11. ATP-Dependent Synthetases and Ligases, 547 Ligation and the Energy of ATP, 547 Activation by Phosphorylation, 548 Glutamine Synthetase, 548 Carbamoyl Phosphate Synthetase, 554 Activation by Adenylylation, 559 DNALigase, 559 Aminoacyl-tRNA Synthetases, 561 Ubiquitin, 566 , Chapter 12. Glycosyl Group Transferases, 569 Chemical Mechanisms, 570 Chemistry of Glycoside Hydrolysis, 570 Enzymatic Glycosyl Transfer, 573 Glycosyltransferases, 575 Sucrose Phosphorylase , 575 Glycogen Phosphorylase, 577 Purine Nucleoside Phosphorylase, 584 Glycosidases, 587 Families and Structures, 587 Lysozyme, 589 T4Lysozyme, 595 Contents Chapter 1 3. Nitrogen and Sulfur Transferases, 597 Nitrogen Transfer, 597 Aspartate Aminotransferase, 597 Tyrosine 2,3-Aminomutase, 602 Amidotransfer, 604 Glutamine:PRPP Amidotransferase, 607 Sulfur Transfer, 609 Biotin Synthase, 611 Lipoyl Synthase, 612 Chapter 14. Carbon-Carbon Condensation and Cleavage, 617 Chemistry, 617 Enolization of Acetyl Co A, 619 Acetyl CoAdn Ester Condensations, 619 Citrate Synthase, 620 Thiolases, 627 Carbanionic Mechanisms, 630 Transaldolase, 631 Transketolase, 634 Serine Hydroxymethyltransferase, 639 Carbocationic Mechanisms, 645 Farnesyl Pyrophosphate Synthase, 645 Squalene Synthase, 648 Chapter 15. Alkyltransferases, 655 Chemistry of Alkylation, 655 Biological Alkylations, 655 Alkylation Mechanisms, 656 Enzymatic Alkylation, 657 Protein Farnesyltransferase, 657 Catechol O-Methyltransferase, 661 S-Adenosylmethionine Synthetase, 665 Methionine Synthases, 670 Chapter 16. Oxidoreductases, 679 Pyridine Nucleotide-Dependent Dehydrogenases, 680 Alcohol Dehydrogenase, 680 Lactate Dehydrogenase, 686 Short-Chain Alcohol Dehydrogenases, 687 Glyceraldehyde-3-P Dehydrogenase, 690 Glutamate Dehydrogenase, 693 Disulfide Oxidoreductases, 694 Dihydrolipoyl Dehydrogenase, 694 Ribonucleotide Reductases, 698 Classes of Ribonucleotide Reductases, 700 Structural Relationships of Ribonucleotide Reductases, 705 xvii xviii Contents Chapter 17. Oxidases and Oxygenases, 710 Oxidases, 710 D-Amino Acid Oxidase, 710 Monoamine Oxidases, 716 Isopenicillin-N Synthase, 718 Urate Oxidase, 721 Monooxygenases, 722 Lactate Monooxygenase, 722 Cytochrome P450 Monooxygenases, 722 Iron-Methane Monooxygenase, 727 oc-Ketoglutarate-Dependent Oxygenases, 732 Dopamine P-Monooxygenase, 735 Copper-Methane Monooxygenase, 737 Nitric Oxide Synthase, 738 Dioxygenases, 741 Intradiol Dioxygenases, 741 Extradiol Dioxygenases, 744 Chapter 18. Complex Enzymes, 749 Multienzyme Complexes, 750 a-Ketoacid Dehydrogenase Complexes, 750 Pyruvate Dehydrogenase Complex, 750 Fatty Acid Synthesis, 757 Acetyl CoA Carboxylase, 757 Fatty Acid Synthases, 761 Modular Enzymes, 763 Polyketide Synthases, 763 Nonribosomal Polypeptide Synthetases, 767 Ribosomal Protein Synthesis, 768 RNA Polymerase, 768 TheRibosome, 770 Energy-Coupling Enzymes, 777 Nitrogenase, 777 Cytochrome c Oxidase, 782 ATP Synthase, 786 Myosin and Muscle Contraction, 792 Appendices, 803 Appendix A: Haldane Relationships for Some Kinetic Mechanisms, 803 Appendix B: Inhibition Patterns for Three-Substrate Kinetic Mechanisms, 804 Appendix C: Equations for Number of Occupied Sites in the Binding of a Ligand to a Multisite Macromolecule, 804 Appendix D: Derivation of Steady-State Kinetic Equations by the King-Altman Method, 805 Index, 809