Enzymes : a practical introduction to structure, mechanism, and data analysis
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書誌事項
Enzymes : a practical introduction to structure, mechanism, and data analysis
Wiley, 2023
3rd ed
- : cloth
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注記
Revised edition of: Enzymes / Robert A. Copeland. 2nd ed. c2000
Includes bibliographical references and index
内容説明・目次
内容説明
ENZYMES A complete and approachable introduction to the study of enzymes, from theory to practice
Enzymes catalyze the bulk of important biological processes, both metabolic and biochemical. They are specialized proteins whose function is determined by their structure, understanding which is therefore a key focus of biological, pharmacological, and agrarian research, among many others. A thorough knowledge of enzyme structure, pathways, and mechanisms is a fundamental building block of the life sciences and all others connected to them.
Enzymes offers a detailed introduction to this critical subject. It analyzes enzyme proteins at the structural level and details the mechanisms by which they perform their catalyzing functions. The book's in-depth engagement with primary literature and up-to-date research allows it to continuously deploy illustrative examples and connect readers with further research on key subjects. Fully updated after decades as the standard text, this book unlocks a thriving field of biological and biochemical research.
Readers of the third edition of Enzymes will also find:
Expanded chapters on steady-state and transient-state enzyme kinetics, structural components of enzymes, and more
New chapters on enzyme regulation, enzyme-macromolecule interactions, enzyme evolution, and enzymes in human health
Key Learning Points at the beginning of each chapter to assist students and instructors
Enzymes promises to continue as the standard reference on this subject for practitioners of the life sciences and related fields in both academia and industry.
目次
Preface to the Third Edition xvii
Preface to the Second Edition xix
Preface to the First Edition xxi
Acknowledgments xxiii
1 A Brief History of Enzymology 1
Key Learning Points 1
1.1 Enzymes in Antiquity 2
1.2 Early Enzymology 3
1.3 The Development of Mechanistic Enzymology 4
1.4 Studies of Enzyme Structure 5
1.5 Enzymology Today 7
1.6 Summary 9
References and Further Reading 9
2 Chemical Bonds and Reactions in Biochemistry 11
Key Learning Points 11
2.1 Atomic and Molecular Orbitals 12
2.1.1 Atomic Orbitals 12
2.1.2 Molecular Orbitals 15
2.1.3 Hybrid Orbitals 16
2.1.4 Resonance and Aromaticity 18
2.1.5 Different Electronic Configurations Have Different Potential Energies 20
2.2 Thermodynamics of Chemical Reactions 22
2.2.1 The Transition State of Chemical Reactions 24
2.3 Acid-base Chemistry 27
2.4 Noncovalent Interactions in Reversible Binding 29
2.4.1 Electrostatic Interactions 30
2.4.2 Hydrogen Bonding 30
2.4.3 Hydrophobic Interactions 31
2.4.4 Van der Waals Forces 31
2.5 Rates of Chemical Reactions 33
2.5.1 Reaction Order 35
2.5.2 Reversible Chemical Reactions 36
2.5.3 Measurement of Initial Velocity 37
2.6 Summary 38
References and Further Reading 38
3 Structural Components of Enzymes 39
Key Learning Points 39
3.1 The Amino Acids 40
3.1.1 Properties of Amino-Acid Side Chains 42
3.1.1.1 Hydrophobicity 42
3.1.1.2 Hydrogen Bonding 42
3.1.1.3 Salt-Bridge Formation 43
3.1.2 Amino Acids as Acids and Bases 44
3.1.3 Cation and Metal Binding 45
3.1.4 Anion and Polyanion Binding 46
3.1.5 Covalent Bond Formation 46
3.1.5.1 Disulfide Bonds 46
3.1.5.2 Phosphorylation 46
3.1.5.3 Glycosylation 47
3.1.6 Steric Bulk 47
3.2 The Peptide Bond 48
3.3 Amino Acid Sequence or Primary Structure 51
3.4 Secondary Structure 54
3.4.1 The Right-Handed 𝛼Helix 55
3.4.2 The 𝛽-Pleated Sheet 56
3.4.3 𝛽Turns 58
3.4.4 Other Secondary Structures 58
3.4.5 Supersecondary Structures 59
3.5 Tertiary Structure 60
3.5.1 Domains 62
3.6 Subunits and Quaternary Structure 64
3.7 Cofactors in Enzymes 67
3.8 Conformational Dynamics and Enzyme Function 70
3.9 Methods of Protein Structure Determination 75
3.9.1 X-ray Crystallography 76
3.9.2 NMR Spectroscopy 77
3.9.3 Cryo-Electron Microscopy (Cryo-EM) 78
3.10 Summary 79
References and Further Reading 80
4 Protein-Ligand Binding Equilibria 83
Key Learnings Points 83
4.1 The Equilibrium Dissociation Constant, Kd 84
4.2 The Kinetic Approach to Equilibrium 86
4.3 Binding Measurements at Equilibrium 88
4.3.1 Derivation of the Langmuir Isotherm 88
4.3.2 Multiple Binding Sites 91
4.3.2.1 Multiple Equivalent Binding Sites 91
4.3.2.2 Multiple Nonequivalent Binding Sites 92
4.3.2.3 Cooperative Interactions Among Multiple Binding Sites 92
4.3.3 Correction for Nonspecific Binding 93
4.4 Graphic Analysis of Equilibrium Ligand-Binding Data 94
4.4.1 Direct Plots on Semilog Scale 94
4.4.2 Linear Transformations of Binding Data: The Wolff Plots 97
4.5 Equilibrium Binding with Ligand Depletion (Tight Binding Interactions) 100
4.6 Competition Among Ligands for a Common Binding Site 101
4.7 Protein Dynamics in Receptor-Ligand Binding 102
4.8 Orthosteric and Allosteric Ligand Binding Sites 104
4.9 Experimental Methods for Measuring Ligand Binding 105
4.9.1 Methods Based on Mass or Mobility Differences 105
4.9.1.1 Equilibrium Dialysis 105
4.9.1.2 Membrane Filtration Methods 107
4.9.1.3 Size Exclusion Chromatography 109
4.9.1.4 Microscale Thermophoresis 111
4.9.2 Spectroscopic Methods 113
4.9.2.1 Fluorescence Spectroscopy 113
4.9.2.2 Surface Plasmon Resonance 116
4.9.3 Ligand-Induced Protein Stabilization 117
4.9.3.1 Thermal Denaturation of Proteins 118
4.9.3.2 Chemical Denaturation of Proteins 120
4.10 Summary 122
References and Further Reading 122
5 Steady-State Kinetics of Single-Substrate Enzyme Reactions 125
Key Learning Points 125
5.1 The Time Course of Enzymatic Reactions 126
5.2 Effects of Substrate Concentration on Velocity 127
5.3 The Rapid Equilibrium Model of Enzyme Kinetics 129
5.4 The Steady-State Model of Enzyme Kinetics 131
5.5 The Significance of kcat and Km 134
5.5.1 Km 135
5.5.2 kcat 135
5.5.3 kcat/Km 136
5.5.4 Diffusion-Controlled Reactions and Kinetic Perfection 138
5.6 Experimental Measurement of kcat and Km 139
5.6.1 Graphical Determinations from Untransformed Data 139
5.6.2 Lineweaver-Burk Plots of Enzyme Kinetics 142
5.7 Other Linear Transformations of Enzyme Kinetic Data 147
5.7.1 Eadie-Hofstee Plots 147
5.7.2 Hanes-Wolff Plots 148
5.7.3 Eisenthal-Cornish-Bowden Direct Plots 148
5.8 Measurements at Low Substrate Concentrations 149
5.9 Deviations From Hyperbolic Kinetics 150
5.10 Summary 153
References and Further Reading 153
6 Chemical Mechanisms in Enzyme Catalysis 155
Key Learning Points 155
6.1 Substrate-Active Site Complementarity 156
6.2 Rate Enhancement Through Transition State Stabilization 159
6.3 Chemical Mechanisms for Transition State Stabilization 162
6.3.1 Approximation of Reactants 163
6.3.2 Covalent Catalysis 166
6.3.2.1 Nucleophilic Catalysis 167
6.3.2.2 Electrophilic Catalysis 168
6.3.3 General Acid/Base Catalysis 170
6.3.4 Conformational Distortion 175
6.3.5 Preorganized Active Site Complementarity to the Transition State 180
6.4 The Serine Proteases: An Illustrative Example 182
6.5 Enzymatic Reaction Nomenclature 187
6.6 Summary 191
References and Further Reading 191
7 Experimental Measures of Steady-State Enzyme Activity 193
Key Learning Points 193
7.1 Initial Velocity Measurements 194
7.1.1 Direct, Indirect, and Coupled Assays 194
7.1.2 Analysis of Progress Curves: Measuring True Steady-State Velocity 200
7.1.3 Continuous Versus End Point Assays 203
7.1.4 Initiating, Mixing, and Stopping Reactions 204
7.1.5 The Importance of Running Controls 206
7.2 Detection Methods 208
7.2.1 Assays Based on Optical Spectroscopy 208
7.2.2 Absorption Measurements 208
7.2.3 Choosing an Analytical Wavelength 210
7.2.4 Optical Cells 210
7.2.5 Errors in Absorption Spectroscopy 212
7.2.6 Fluorescence Measurements 213
7.2.7 Internal Fluorescence Quenching and Energy Transfer 215
7.2.8 Errors in Fluorescence Measurements 217
7.2.9 Radioisotopic Measurements 220
7.2.10 Errors in Radioactivity Measurements 223
7.2.11 Other Detection Methods 223
7.3 Separation Methods in Enzyme Assays 224
7.3.1 Separation of Proteins from Low Molecular Weight Solutes 224
7.3.2 Chromatographic Separation Methods 225
7.3.3 Electrophoretic Methods in Enzyme Assays 230
7.4 Factors Affecting the Velocity of Enzymatic Reactions 236
7.4.1 Enzyme Concentration 237
7.4.2 pH Effects 239
7.4.3 Temperature Effects 245
7.4.4 Viscosity Effects 247
7.4.5 Isotope Effects in Enzyme Kinetics 249
7.5 Reporting Enzyme Activity Data 252
7.6 Enzyme Stability 253
7.6.1 Stabilizing Enzymes During Storage 254
7.6.2 Enzyme Inactivation During Activity Assays 255
7.7 Summary 258
References and Further Reading 258
8 Transient-State Kinetics 261
Key Learning Points 261
8.1 Timescale of Pre-Steady-State Turnover 262
8.2 Instrumentation for Transient Kinetic Measurements 264
8.3 Estimating Initial Conditions for Transient Kinetic Measurements 266
8.4 Examples of Some Common Transient Kinetic Reaction Mechanisms 267
8.4.1 One Step, Irreversible Binding 267
8.4.2 One Step, Reversible Binding 268
8.4.3 Consecutive, Irreversible Reaction 268
8.4.4 Consecutive, Reversible Reaction with a Fast First Step (Pre-equilibrium Reaction) 269
8.4.5 Consecutive, Reversible Reaction with a Fast Second Step (Enzyme Pre-isomerization) 271
8.5 Examples of Transient Kinetic Studies from the Literature 272
8.5.1 Study of Substrate and Inhibitor Interactions with the Alzheimer's Disease -Site Amyloid Precursor Protein-Cleaving Enzyme (BACE) 272
8.5.2 Study of the Mechanism of Time-Dependent Inhibition of Staphylococcus aureusPolypeptide Deformylase 275
8.6 Summary 277
References and Further Reading 278
9 Enzyme Regulation 279
Key Learning Points 279
9.1 Active and Inactive Conformational States 280
9.2 Post-Translational Modifications 281
9.2.1 Proteolytic Processing 282
9.2.2 Covalent Modification of Amino Acid Side Chains 288
9.3 Enzyme Regulation Through Protein-Protein Interactions 294
9.4 Small-Molecule Allosteric Ligands 297
9.4.1 Homotropic and Heterotropic Allostery 298
9.4.2 Intramolecular and Intermolecular Allostery 298
9.5 Quantitative Measurements of Enzyme Activation and Inhibition 302
9.5.1 Thermodynamic Measurement of Activator-Enzyme Interactions 303
9.5.2 Kinetic Measurement of Enzyme Activation by PTM 306
9.6 Regulation of Protein Kinases 308
9.6.1 Kinase Activation by PTM 308
9.6.2 Kinase Regulation by Protein Association 311
9.6.3 Kinase Activation by Oligomerization 312
9.6.4 Kinase Regulation by Small-Molecule Binding 313
9.6.5 Small-Molecule Mimicry of Intramolecular Allostery 313
9.7 Summary 314
References and Further Reading 315
10 Reversible Inhibitors 317
Key Learning Points 317
10.1 Equilibrium Treatment of Reversible Inhibition 319
10.2 Thermodynamic Modes of Reversible Inhibition 321
10.2.1 Pure Competitive Inhibition, Exclusive Binding to Free Enzyme (E): 𝛼= 321
10.2.2 Mixed or Noncompetitive Inhibition 322
10.2.2.1 Mixed Inhibitors That Bind Preferentially to the Free Enzyme (E): 𝛼 >1 322
10.2.2.2 Mixed Inhibitors That Bind Equipotently to E and ES: 𝛼=1 322
10.2.2.3 Mixed Inhibitors That Bind Preferentially to the Enzyme-Substrate Complex (ES): 𝛼 <1 322
10.2.3 Pure Uncompetitive Inhibitors, Exclusive Binding to the Enzyme-Substrate Complex (ES): 𝛼 1 323
10.2.4 Partial Inhibitors 323
10.3 Effects of Inhibitors on Steady-State Parameters 324
10.3.1 Competitive Inhibitors 325
10.3.2 Noncompetitive Inhibitors 329
10.3.3 Uncompetitive Inhibitors 330
10.3.4 Fitting of Untransformed Data 332
10.4 Concentration-Response Plots of Enzyme Inhibition 333
10.4.1 Concentration-Response Plots for Partial Inhibition 336
10.5 Effects of Substrate Concentration on Inhibitor Concentration-Response Curves 337
10.6 Mutually Exclusive Binding of Two Inhibitors 340
10.7 Structure-Activity Relationships and Inhibitor Design 343
10.7.1 SAR in the Absence of Enzyme Structural Information 343
10.7.2 Inhibitor Design Based on Enzyme Structure 350
10.8 Summary 353
References and Further Reading 354
11 Tight-Binding Inhibitors 357
Key Learning Points 357
11.1 Identifying Tight-Binding Inhibition 358
11.2 Distinguishing Inhibitor Type for Tight-Binding Inhibitors 359
11.3 Determining Ki for Tight-Binding Inhibitors 362
11.4 Use of Tight-Binding Inhibitors to Determine Active Enzyme Concentration 365
11.5 Summary 368 References and Further Reading 368
12 Time-Dependent Inhibition 371
Key Learning Points 371
12.1 Progress Curves for Slow-Binding Inhibitors 375
12.2 Distinguishing Between Slow-Binding Schemes 378
12.2.1 Scheme B 379
12.2.2 Scheme C 379
12.2.3 Scheme D 380
12.3 Distinguishing Between Modes of Inhibitor Interaction with Enzyme 382
12.4 Determining Reversibility 384
12.4.1 Enzyme-Inhibitor Residence Time 385
12.5 Examples of Slow-Binding Enzyme Inhibitors 386
12.5.1 Serine Proteases 386
12.5.2 Prostaglandin G/H Synthase 387
12.5.3 Chemical Modification as Probes of Enzyme Structure and Mechanism 391
12.5.3.1 Amino Acid Selective Chemical Modification 392
12.5.3.2 Substrate Protection Experiments 394
12.5.3.3 Affinity Labels 396
12.6 Summary 398
References and Further Reading 398
13 Enzyme Reactions with Multiple Substrates 401
Key Learning Points 401
13.1 Reaction Nomenclature 402
13.2 Bi-Bi Reaction Mechanisms 403
13.2.1 Random Ordered Bi-Bi Reactions 403
13.2.2 Compulsory-Ordered Bi-Bi Reactions 404
13.2.3 Double Displacement or Ping-Pong Bi-Bi Reactions 406
13.3 Distinguishing Between Random and Compulsory-Ordered Mechanisms by Inhibition Pattern 407
13.4 Isotope Exchange Studies for Distinguishing Reaction Mechanisms 409
13.5 Using the King-Altman Method to Determine Velocity Equations 411
13.6 Cleland's Net Rate Constant Method for Determining Vmax and Vmax/Km 414
13.7 Summary 416
References and Further Reading 417
14 Enzyme-Macromolecule Interactions 419
Key Learning Points 419
14.1 Mutlitprotein Enzyme Complexes 420
14.2 Enzyme Reactions on Macromolecular Substrates 422
14.2.1 Differences Between Small Molecule and Protein Substrate Binding to Enzymes 422
14.2.2 Factors Affecting Protein-Protein Interactions 425
14.2.3 Separation of Binding and Catalytic Recognition Elements 427
14.2.4 Noncompetitive Inhibition by Active Site Binding Molecules for Exosite Utilizing Enzymes 429
14.2.5 Processive and Distributive Mechanisms of Catalysis 430
14.2.6 Effect of Substrate Conformation on Enzyme Kinetics 434
14.2.7 Inhibitor Binding to Substrates 434
14.3 Summary 436
References and Further Reading 436
15 Cooperativity in Enzyme Catalysis 439
Key Learning Points 439
15.1 Historic Examples of Cooperativity and Allostery in Proteins 441
15.2 Models of Allosteric Behavior 445
15.3 Effects of Cooperativity on Velocity Curves 449
15.4 Sigmoidal Kinetics for Nonallosteric Enzymes 452
15.5 Summary 453
References and Further Reading 453
16 Evolution of Enzymes 455
Key Learning Points 455
16.1 Early Earth Conditions 456
16.2 Natural Selection 456
16.3 Genetic Alterations 459
16.3.1 Single Nucleotide Polymorphisms (SNPs) 459
16.3.2 Gene Duplication 460
16.3.3 Deletions and Insertions 461
16.3.4 Translocations and Inversions 461
16.4 Enzyme Families and Superfamilies 463
16.5 Enzyme Promiscuity as a Springboard of Evolution 467
16.5.1 Evolution of Enzyme Steady State Parameters 471
16.6 Protein Dynamics and Conformational Selection in Evolution of Neofunctionality 474
16.7 Ancestral Enzyme Reconstruction 475
16.7.1 Mechanism of Drug Selectivity for Gleevec 477
16.7.2 Overcoming Epistasis to Define the Mechanism of Substrate Specificity 478
16.7.3 Revealing Generalist to Specialist Evolution 479
16.7.4 Ancestral Sequence Reconstruction as a Practical Tool 480
16.8 Contemporary Enzyme Evolution 480
16.9 Summary 483
References and Further Reading 483
17 Enzymes in Human Health 487
Key Learning Points 487
17.1 Enzymes as Therapeutic Agents 487
17.2 Enzyme Inhibitors as Therapeutic Agents 488
17.2.1 Properties of Small-Molecule Drugs 489
17.2.2 Enzymes as Drug Targets 489
17.3 Enzyme Essentiality in Disease 492
17.3.1 Paralogues with Distinct Physiological Roles 492
17.3.2 Distinct Orthologues in Infectious Diseases 494
17.3.3 Diseases of Lifestyle, Environmental, and Aging 497
17.3.4 Pathogenic Alterations to Enzyme Function 501
17.3.4.1 Relating Genetic Alterations to Disease Essentiality 502
17.3.4.2 Enzyme Overexpression 505
17.3.4.3 Activating Mutations 506
17.3.4.4 Chromosomal Translocations 515
17.3.4.5 Synthetic Lethality 518
17.3.5 Pro-Drug Activation by Enzymes 522
17.4 Enzyme-Mediated Target Protein Degradation 524
17.5 The Role of Enzymology in Drug Discovery and Development 527
17.5.1 Enzyme Selectivity Assessment 529
17.5.2 Correlating Enzyme Inhibition with Cellular Phenotype 530
17.5.3 Hepatic Metabolism of Xenobiotics 533
17.5.4 Mutation-Based Drug Resistance 535
17.6 Summary 537
References and Further Reading 537
Index 543
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