Enzymes (3° Éd.) A Practical Introduction to Structure, Mechanism, and Data Analysis

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.

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, K_d 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 k_cat and Km 134

5.5.1 K_m 135

5.5.2 k_cat 135

5.5.3 k_cat/K_m 136

5.5.4 Diffusion-Controlled Reactions and Kinetic Perfection 138

5.6 Experimental Measurement of k_cat and K_m 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 β-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 V_max and V_max/K_m 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

Robert A. Copeland, PhD, is founder, President, and Chief Scientific Officer (CSO) of Accent Therapeutics, Inc. and the President of Ki Consultant, LLC. Previously, he was President of Research and CSO of Epizyme, Inc., and Vice President for Cancer Biology at the Oncology Center of Excellence in Drug Discovery for GlaxoSmithKline. He is a fellow of the American Association for the Advancement of Science and the Royal Society of Chemistry, and has published very widely on enzymes and related subjects.