Enzymatic reaction mechanisms /

Chapter 1. ENZYMES AND CATALYTIC MECHANISMS 1
Catalysis and the active site
Rate enhancement in enzymatic catalysis 3
Conformational mobility in catalysis 5
Substrate-induced conformational changes 6
Catalysis of multistep reactions 7
Structural mobility in enzymes 8
Quenching of tryptophan fluorescence by O2 8
Packing densities 9
Nature of motions 10
Time regimes of motions 11
Acid-base catalysis 12
Acids and bases 12
Specific acid-base catalysis 13
General acid-base catalysis 15
Concerted acid-base catalysis 19
Bronsted catalysis law 20

Nucleophilic catalysis 22
Electrophilic catalysis 26
Catalysis of enolization 27
Imine formation by lysine 29
Mechanism of imine formation 29
Aldolase 32
Catalysis by metal ions 33
Class II aldolases 34
Enolase 35

Hydrogen bonding 36
Strong and weak hydrogen bonds 37
Hydrogen bonding in catalysis 41
Binding energy in catalysis 43
Binding and activation energy 43
The active site as an entropy trap 46
Intramolecular reactions 46
Loss of translational and rotational entropy 48
Dissecting the binding effect in enzymatic action 50
Stabilization of the transition state 51
The transition state theory 52
Enzymatic rate enhancement and transition state binding 53
Cytidine deaminase 55
vi
Binding near attack conformations 59
Destabilization of the ground state 60
Rate enhancement through binding of remote groups 60
The transition state defined 60
Acetoacetyl CoA:succinate CoA transferase 62
Characterization of active sites 66
Competitive inhibitors¿Analogs of substrates 66
Group selective chemical modification 66
Identification of essential emino acids 67
Glu35 and Asp52 in lysozyme 68
Site-directed mutagenesis 70
Complementarity of site-directed mutagenesis and
Group selective chemical modification 70
Proof of function 72
Kinetic consequences of mutating a catalytic residue 72
Affinity Labeling 73
Kinetics 74
Chymotrypsin and TPCK 75
Why are enzymes large molecules? 78
Sizes of enzymatic binding domains 78
Catalytic antibodies 79
Transesterification 80
Scope and limitations 81
Chapter 2 KINETICS OF ENZYMATIC 89
Steady-State Kinetics 89
One-Substrate Reactions 90
Two-substrate Reactions 96
Sequential binding mechanisms 98
Adenylate kinase 100
Ordered sequential mechanisms 101
Ping pong mechanisms 103
Nucleoside diphosphate kinase 104
Two-site ping pong mechanisms 108
Haldane relationships 109
Inhibition patterns 110
Isotope exchange at equilibrium 116.
Three-substrate reactions 117
General rate law 117
Examples 118
Inhibition patterns 120
vii
Isotope Effects 120
Classes of isotope effects 120
Kinetic and equilibrium isotope effects 120
Spectroscopic isotope effects 121
Magnitudes of kinetic isotope effects 122
Measurement of isotope effects 126
Direct method 126
Internal competition 126
Remote label 127
Equilibrium perturbation 128
Intrinsic isotope effect 129
Hydrogen tunneling 132
Transient Phase Kinetics 135
Transient methods 136
Burst kinetics 136
Stopped flow spectrophotometry 139
Rapid mix-quench 140
Kinetics of partial reactions:
Tyrosyl-tRNA synthetase 141
Nonsteady state analysis 142
Relaxation methods 143
Estimation of off-rates by
isotope partitioning 145

pH-Rate Profiles 147
Measurement of pH-profiles 147
Parameters of interest 147
pH-Dependence of Ki 147
pH-Dependence of V and V/K 149
¿Indistinguishable¿ values of pKa 151
Reverse protonation 152
Assignment of pKa in enzymes 152
The pH-dependence of chymotrypsin 153
Allosteric Regulation 154
Theory 154
Concerted and ligand-induced
transition models 156
A general theory of cooperativity 157
Binding equations for cooperative systems 158
Ligand binding to a multisite macromolecule 158 The Hill equation 160
viii
Aspartate transcarbamoylase 161
Kinetics 161
Structure 163
Reaction mechanism 163

Chapter 3 COENZYMES I: ORGANIC COENZYMES 169
Nicotinamide Coenzymes 169
Structures and functions of nicotinamide coenzymes 169
Stereospecifity of hydride transfer 172
NAD+ as a coenzyme 173
UDP-galactose 4-epimerase 174
Nucleotide sugar 4,6-dehydratases 174
S-Adenosylhomocysteine hydrolase 175
Dehydroquinate synthase 176
Ornithine cyclodeaminase 177
Urocanase 178

Thiamine Pyrophosphate 178
Structure and reactions 178
Phosphoketolase 181
Pyruvate oxidase 181
Pyruvate oxidoreductases 183
?-Lipoamide 184
Pyridoxal 5'-Phosphate 185
Enzymatic reactions facilitated by PLP 185
PLP-stabilized amino acid carbanions 186
Mechanisms of PLP-dependent reactions 187
Tryptophan synthase 189
Role of PLP in radical isomerizations 191
Flavin Coenzymes 192
Structures of flavin coenzymes 192
Mechanisms of flavin catalysis 193
Flavin-dependent oxidoreductases 194
Flavoprotein oxidases 195
Biopterin 195
Biotin 196
Structure and role as a carboxyl carrier 196
Chemistry of biotin and N1-carboxybiotin 197
Mechanism of biotin-dependent carboxylation 198

ix
Phosphopantetheine Coenzymes 199
Structures of phosphpantetheine coenzymes 199
Mechanism of phosphopantetheine-action 200
The chemical role 200
The physical role 201

Folate Coenzymes 201
Folate compounds of one-carbon metabolism 202
Enzymes in H4-folate metabolism 203
Serine hydroxymethyltransferase 203
Thymidylate synthase 203
Formyl-H4folate-dependent formyl transferases 204
Biological importance of folate 204
Amino acid-based coenzymes 205
Pyruvoyl decarboxylases 205
Methylidene Imidazolone-dependent deaminases 206
Quinoproteins 207
Pyrroloquinoline quinone (PQQ) 208
Tryptophan tryptophyl quinone (TTQ) 210
Topaquinone (TPQ) 211
Chapter 4. COENZYMES II: METALLIC COENZYMES 221
Vitamin B12 Coenzymes 222
Chemistry of B12 Coenzymes 222
Structures 222
Chemical properties of B12 compounds 223
Adenosylcobalamin-dependent enzymes 224
Isomerization reactions 225
Adenosylcobalamin and hydrogen transfer 226
Chemical models for radical isomerizations 230
Ribonucleotide reductase 230
Methylcobalamin-dependent enzymes 233
Methionine synthase 234
Methylcoenzyme M synthase 235
Heme Coenzymes 235
Chemistry of oxygen and heme 235
Oxygen structure and chemistry 235
Structure and chemistry of heme 238
Heme enzymes 240
Catalase 240
Peroxidases 241
Cytochrome P450s 242
x
Oxygen binding and electron transfer 243
Myoglobin and Hemoglobin 243
Cytochrome c 244
Mononuclear non-heme iron 244
Monooxygenases 246
Phenylalanine hydroxylase 246
?-Ketoglutarate as reductant 252
Dioxygenases 252

Oxo-Fe2 complexes 253
Structures 253
Reactions of di-iron enzymes 254
Hydrogen atom abstraction 255
Fatty acid desaturases 255
Purple acid phosphatases 258
Metallopterin enzymes 255
Molybdopterin and Tungstopterin 259
Coenzyme structures 259
Xanthine oxidase 260
Sulfite oxidase family 262
Dimethylsulfoxide reductase 263
Iron-Sulfur Centers 264
Structures 265
Catalytic functions 266
Electron Transfer 266
Aconitase and ???-dehydratases 267
???-dehydratases 269
Molybdenum-iron-sulfur center of nitrogenase 270
S-Adenosylmethionine/[4Fe¿4S] 270
Catalytic action of SAM/[4Fe¿4S] 271
Lysine 2,3-aminomutase 271
Spore photoproduct lyase 273
Stoichiometric reactions of SAM/[4Fe¿4S] 273
Divalent Metal Ions 274
Electrostatic activation of coordinated water 274
Electrostatic activation of enolization. 275
xi
Copper as a cofactor 276
Copper proteins 277
Ascorbate oxidase 277
Galactose oxidase 278
Other copper enzymes 279
Nickel Coenzymes 279
Nickel in methanogenesis 280
Methanogenesis 280
Methyl coenzyme M reductase 280
Other nickel coenzymes 282
Long Range Electron Transfer 282
Biological electron transfer 282
Marcus theory 284
The Marcus equation 284
Electron tunneling 285
Natural selection in electron transfer? 286
Chapter 5 ENZYME INHIBITION 295

Two-Substrate Analogs 296
PALA and aspartate carbamyltransferase 296
Suicide Inactivation 297
Thymidylate synthase 298
5-Fluoro-dUMP 298
Reaction and molecular properties 299
Reaction mechnanism 299
Inactivation by FdUMP 301
Structure 302
3-Hydroxydecanoyl CoA Dehydratase 304
Reaction and mechanism 304
Acetylenic inactivator 305
GABA Aminotransferase 306
Gabaculine 306
?-Vinyl-GABA 307
Structure 308
Kinetics of suicide inactivation 309
Fluorinated analogues of GABA 310
Kinetics of Slow and Tight Binding Inhibition 311
Slow binding 312
Tight binding 314
xii
Slow Binding Inhibition 316
Dihydrofolate reductase 317
Function 317
Methotrexate 317
Structure 318
Kinetics 319
Structural dynamics 320
Prostaglandin H synthase 320
Function 320
Structure 321
Reaction mechanism 322
Slow and fast binding COX inhibitors 324
Tight Binding Inhibition 325
HMG-CoA reductase 325
Properties and function 325
Reaction mechanism 326
Inhibition by statins 327
Molecular structure 328
Alanine racemase 329
Function and properties 329
Reaction mechanism 330
Inhibition by 1-aminoethylphosphonate 332
Molecular structure 332
Enolpyruvoylshikimate Synthase 334
Function and mechanism of action 334
Glyphosate 334
Acetylcholinesterase 335
Biological function 335
Inhibitors 336
Fasciculin 337
Reaction mechanism 337
Chapter 6 ACYL GROUP TRANSFER:PROTEASES 346
Chemistry of acyl transfer 346
Serine proteases 350
Chymotrypsin 351
Transient and steady state kinetics 352
Partitioning of the acyl-chymotrypsin 353
Acyl-chymotrypsin and Ser195 354
pH-Dependence and His57 354
Role of Ile16 355
Kinetic isotope effects 357
xiii
Ser195 adducts 360
The oxyanion site 362
The low-barrier hydrogen bond 363
Subtilisin 365
Convergent evolution 365
Chemical mutagenesis 366
Engineered subtilisin 366
The LBHB 367
Cysteine proteases 368
Papain 368
Kinetics and isotope effects 369
Structure 369
Roles of Cys25 and His159 370
Caspases 371
Aspartate proteases 371
Molecular properties 372
Pepsin 372
HIV protease 373
Mechanism of action 374
An acid/base mechanism 375
The iso-mechanism 375
The postulated LBHB 376
Metalloproteases 377
Carboxypeptidase A 377
Kinetics 378
Structure 380
Inhibition by peptide phosphonates 380
Thermolysin 381
Esterases 381
Phospholipase A2 382
Chapter 7 ISOMERIZATION 391
Aldose-Ketose Isomerases 391
Chemistry 391
Phosphoglucose isomerase 392
Triosephosphate isomerase 394
Xylose isomerase 400
xiv
Phosphomutases 401
?-Phosphoglucomutase 401
?-Phosphoglucomutase 403
Phosphoglycerate mutases 404

Epimerases and Racemases 407
Proline racemase 407
Glutamate racemase 411
Ionization of amino acid ?-protons 412
Mandelate racemase 413
UDP-galactose 4-epimerase 417
Dehydrogenation at glycosyl-C4 418
Nonstereospecificity 419
Mechanism of hydride transfer 420
Uridine nucleotide-induced reductive inactivation 421
Ribulose-5-phosphate 4-epimerase 422
UDP-N-acetylglucosamine 2-epimerase 424
Chorismate Mutase 426
?5-3-Ketosteroid isomerase 428
Radical Isomerizations 430
Glutamate mutase 430
Methylmalonyl-CoA mutase 433
Lysine 2,3-aminomutase 438
Newer Isomerases 441
UDP-galactopyranose mutase 441
Pseudouridine synthase 443
Chapter 8 DECARBOXYLATION AND CARBOXYLATION 455
Chemistry of Decarboxylation and Carboxylation 455
Decarboxylases 456
Pyruvate Decarboxylase 457
TPP-intermediates 457
The structure of yeast PDC 459
Mechanism of catalysis 460
Amino acid Decarboxylases 462
Dialkylglycine decarboxylase 463
Aspartate??-decarboxylase 465
Histidine decarboxylase 466
Acetoacetate Decarboxylase 469
Mevalonate-PP Decarboxylase 472
xv
Radical-based Decarboxylases 474
Pyruvate-Formate Lyase (PFL) 474
PFL Activase 478
Coproporphyrinogen Oxidases 478
Oxalate Decarboxylase 479
Orotidine Monophosphate Decarboxylase 481
Carboxylases 485
Ribulose Bisphosphate Carboxylase 486
Reaction and properties 486
Activation 488
Mechanism of action 488
Phosphoenolpyruvate Carboxylase 491
Vitamin K-dependent carboxylase 493
Chapter 9 ADDITION and ELIMINATION 505
???-Elimination/Addition 505
Cofactor-independent ???-elimination/addition 505
Fumarase 506
Enoyl-CoA hydratase 509
Cofactor-dependent????-elimination/addition 513
Enolase and the role of Mg2+ 513
Aconitase and the role of [4Fe¿4S] 515
dTDP-glucose 4,6-dehydratase and the role of NAD+ 520
PLP-dependent ???-elimination 523
PLP-dependent ?-replacement 524
PLP-dependent ???-elimination 527
???-Elimination/Addition 528
Methylidene imidazolone-dependent 529
Histidine ammonia-lyase 529
Phenylalanine ammonia-lyase 531
Carbonic Anhydrase 534
Isomerization/Elimination 538
Coenzyme B12-dependent elimination 538
Dioldehydrase 538
Ethanolamine ammonia-lyase 544
xvi
Chapter 10 PHOSPHOTRANSFER AND NUCLEOTIDYLTRANSFER 551
Chemistry of phosphoryl group transfer 551
Phosphmonoesters 551
Transition state for phosphoryl group transfer 551
pH-Dependence of hydrolysis 552
Electronic effects on hydrolysis 553
Activation parameters and solvent partitioning 554
Special cases of hydrolysis 555
18O-Kinetic isotope effects 556
Phosphoryl group transfer to nucleophiles
other than solvent 557
Stereochemistry 559
Discrete metaphosphate anion? 559
Phosphodiesters 561
Phosphotriesters 562
Five-member ring phosphoesters 563
Enzymatic phosphoryl group transfer 565
Single and Double Displacements 565
Steady state kinetics 566
Stereochemistry 567
Characterization of covalent phosphoenzymes 568
Phosphotransferases 570
Adenylate kinase and nucleoside diphosphate kinase 570
Creatine kinase 571
Acetate kinase 574
Phosphoglycerate kinase 575
Pyruvate kinase 576
Pyruvate Phosphate Dikinase 580
Phosphofructokinase 581
Protein phosphorylation: Protein kinase A 584
Action of the catalytic subunit 587
Structure-function in a protein tyrosine kinase 590
Action of the regulatory subunit 591
Protein kinase inhibitor and A-kinase anchoring proteins 593
Phosphomonoesterases 593
Histidine phosphatases 595
Cysteine phosphatases 598
Serine phosphatases 602
Metallophosphatases/pyrophosphatase 605
xvii
Enzymatic nucleotidyl group transfer 607
Nucleotidyltransferases 607
Chemistry of nucleotidyl transfer 607
Adenylyl cyclase 608
Uridylyltransferases 612
DNA Polymerase 615
Topoisomerase 618
Ribozymes 620
Phosphodiesterases 623
Ribonuclease 623

Chapter 11 ATP-DEPENDENT SYNTHETASES AND LIGASES 639
Ligation and the Energy of ATP 639
Activation by Phosphorylation 640
Glutamine Synthetase 640
Reaction and energetics 640
Kinetic mechanism 641
Chemical mechanism 642
Regulation in bacteria 643
Inhibition 643
Structure 644
Carbamoyl Phosphate Synthetase 645
Chemical mechanism 646
Kinetics 647
Structure 649
Activation by Adenylylation 650
DNA Ligase 651
Chemical mechanism 651
Kinetic mechanism 652
Structure of ATP-dependent ligase 653
Aminoacyl tRNA Synthetases 653
Translation 653
Classes of synthetases 654
Mechanism of action 655
Fidelity of action 656
Ubiquitin 659
xviii
Chapter 12 GLYCOSYL GROUP TRANSFERASES 665
Chemical Mechanisms 665
Chemistry of glycoside hydrolysis 665
Acid catalysis 665
2-Acetamido participation-Anchimeric assistance 667
Transition state 668
Enzymatic glycosyl transfer 670
Retaining and inverting enzymes 670
Generic mechanisms 671
Glycosyltransferases 671
Sucrose phosphorylase 672
Reaction, stereochemistry, and kinetics 672
Glucosyl¿enzyme intermediate 673
Glycogen phosphorylase 675
Reaction and stereochemistry 675
Regulation 676
Kinetics 677
Structure 679
PLP and the mechanism of glycosyl transfer 680
Mechanism of action 681
Purine nucleoside phosphorylase 683
Reaction, stereochemistry, and kinetics 683
Isotope effects and the transition state 684
Immucillin-H, a transition state analogue 685
Glycosidases 686
Families and structures 686
Lysozyme 687
Reaction and structure 687
Substrate distortion 689
Isotope and pH-effects 690
The glycosyl-enzyme intermediate 691
Transition state 692
T4 Lysozyme 693


Chapter 13. NITROGEN AND SULFUR TRANSFERASES 698
Nitrogen transfer 698
Aspartate aminotransferase 698
Reaction and kinetics 698
Structure 700
Catalytic mechanism 701
xvix
Tyrosine 2,3-aminomutase 703
Amidotransfer 704
Glutamine as a source of NH3 704
Diverse mechanisms in reactions of NH3 705
Glutamine:PRPP amidotransferase 706
Two classes of amidotransferase 707
Glutaminase action 707
Structure 694
Regulation¿end-product inhibition 709
Sulfur transfer 710
Biotin synthase 710
Reaction and stoichiometry 710
SAM and iron-sulfur centers 711
Structure and reaction mechanism 712
Lipoyl synthase 713
Chapter 14 CARBON-CARBON CONDENSATION AND CLEAVAGE 719
Chemistry 719
Enolization of acetyl-CoA 721
Acetyl-CoA in ester condensations 721
Citrate synthase 721
Molecular properties and regulation 721
Stereochemistry 722
Reaction mechanism 725
Structure 727
Enolization mechanism 727
?hiolases 731
Degradative and biosynthetic thiolases 731
Kinetics and mechanism 732
Structure of acetoacetyl-CoA thiolase 734
Carbanionic mechanisms 735
Transaldolase 736
Reaction and kinetics 736
Structure 737
Reaction mechanism 738
Transketolase 739
Reaction and kinetics 739
Structure 740
Reaction mechanism 741
xx
Serine Hydroxymethyltransferase 742
Reactions and metabolic role 742
Molecular properties 743
Active site structure 743
Reaction mechanism 744
Carbocationic mechanisms 748
Farnesyl pyrophosphate synthase 749
Reaction and stereochemistry 749
Condensation mechanism 749
Structure and mutagenesis 751
Squalene Cyclase 752
Chapter 15 ALKYLTRANSFERASES 760
Chemistry of alkylation 760
Biological alkylations 760
Alkylation mechanisms 761
Enzymatic alkylation 763
Protein farnesyltransferase 763
Reaction 763
Role of zinc 764
Kinetics 764
Structure 765
Reaction mechanism 767
Catechol O-methyltransferase 767
Chemistry of methyl transfer 767
Properties of COMT 769
Reaction of COMT 769
Structure 770
Reaction mechanism 771
S-Adenosylmethionine synthetase 772
Reaction and kinetics 772
Structure and mechanism of action 773
Methionine synthases 776
Reaction and properties of MetH 776
Modular structure 778
Modular function 781
Reaction mechanisms in catalysis and repair 782
xxi
Chapter 16 OXIDOREDUCTASES 789
Pyridine Nucleotide-Dependent Dehydrogenases 790
Alcohol dehydrogenase 790
Reaction, stereochemistry, and kinetics 791
Structure 792
Role of zinc 794
Transient kinetics 795
Isotope effects 797
Catalysis of hydride transfer 797
Lactate dehydrogenase 798
Reaction, stereochemistry, and molecular properties 798
Structure and reaction mechanism 799
Short chain alcohol dehydrogenases 800
Glyceraldehyde-3-P dehydrogenase 801
Reaction and stereospecificity 801
Kinetics 802
Binding of NAD+ 803
Structure 803
Glutamate dehydrogenase 804
Disulfide Oxidoreductases 805
Dihydrolipoyl dehydrogenase 806
Reaction and kinetics 806
Structure and reaction mechanism 807
Covalent addition to FAD 809

Ribonucleotide reductases 810
Classes of reductases 812
Class I RNR 813
Class II RNR 815
Class III RNR 816
Structural relationships in reductases 818
Chapter 17 OXIDASES AND OXYGENASES 827
Oxidases 827
D-Amino acid oxidase 827
Reaction and properties 827
Hypothetical mechanisms of flavin reduction 828
xxii
Monoamine oxidases 832
Reaction mechanism 832
Structure of MAO B 833
Isopenicillin N synthase 834
Urate oxidase 836
Monooxygenases 837
Lactate monooxygenase 837
Cytochrome P450 monooxygenases 837
Hydroxylation of alkanes 837
Hydroxylation cycle 838
Oxygenation mechanism 839
Structure of P450 cam 841
Iron-Methane monooxygenase 843
Hydroxylation of substrates 843
Structure 844
Hydroxylation cycle 844
Oxygenation mechanism 845
?-Ketoglutarate-dependent monooxygenases 848
?-Ketoglutarate as a reducing system 848
Deacetoxycephalosporin C synthase 849
Dopamine ?-monooxygenase 850
Reaction and molecular properties 851
Kinetics and isotope effects 851
Reaction mechanism 852
Copper-Methane monooxygenase 853
Nitric oxide synthase 855
Reaction and molecular properties 855
Structure 856
Aspects of reaction mechanism 856
Dioxygenases 857
Intradiol dioxygenases 857
Protocatachuate 3,4-dioxygenase 857
Extradiol dioxygenases 859

CHAPTER 18 COMPLEX ENZYMES 868
Multienzyme complexes 869
??Ketoacid dehydrogenase complexes 869
xxiii
Pyruvate dehydrogenase complexes 869
Stepwise reaction mechanism 869
Composition and structure¿E1, E2, E3 870
TPP in decarboxylation and electron transfer 872
Site-site interactions in the E1 dimer 872
Reductive acylation of lipoamide 873
Fatty acid synthesis 875
Acetyl CoA carboxylase 875
Reaction and molecular properties 875
Carboxylation mechanism 877
Structure 878
Fatty acid synthase 879
Processing acetyl and malonyl CoA into palmitate 879
Multienzyme fatty acid synthase systems 880
Modular enzymes 881
Polyketide synthases 881
Type I PKS 881
Type II PKS 883
Type III PKS and others 884
Nonribosomal polypeptide synthases 884
ACV synthetase 885
Ribosomal polypeptide synthesis 886
RNA polymerase 886
The ribosome 888
Structure 888
Initiation 889
Elongation 890
Termination 891
Peptide formation 892
Energy coupling enzymes 893
Nitrogenase 894
Reactions of nitrogenase 894
Structure 895
ATP-dependent electron transfer 897
Mechanism of N2-reduction 898
Cytochrome oxidase 899
Function and molecular composition of CcO 900
Structure of CcO 901
Reduction of O2 902
Proton pumping 903
xxiv
ATP synthase 904
Molecular properties 904
Chemistry and the binding change mechanism 906
Molecular structure 909
Rotatory action 910
Myosin and muscle contraction 910
The sliding filament model 910
Structures of myosin heads 911
Kinetics of myosin action 912
The myosin-actin interaction 913
Mechanochemical coupling 915