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Enzymes. A Practical Introduction to Structure, Mechanism, and Data Analysis. Edition No. 3

  • Book

  • 576 Pages
  • February 2023
  • John Wiley and Sons Ltd
  • ID: 5837027
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.

Table of Contents

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

2.2 Thermodynamics of Chemical Reactions 22

2.3 Acid-base Chemistry 27

2.4 Noncovalent Interactions in Reversible Binding 29

2.5 Rates of Chemical Reactions 33

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

3.2 The Peptide Bond 48

3.3 Amino Acid Sequence or Primary Structure 51

3.4 Secondary Structure 54

3.5 Tertiary Structure 60

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

3.10 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

4.4 Graphic Analysis of Equilibrium Ligand-Binding Data 94

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

4.10 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 K m 134

5.6 Experimental Measurement of k cat and K m 139

5.7 Other Linear Transformations of Enzyme Kinetic Data 147

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

6.4 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

7.2 Detection Methods 208

7.3 Separation Methods in Enzyme Assays 224

7.4 Factors Affecting the Velocity of Enzymatic Reactions 236

7.5 Reporting Enzyme Activity Data 252

7.6 Enzyme Stability 253

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

8.5 Examples of Transient Kinetic Studies from the Literature 272

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

9.3 Enzyme Regulation Through Protein-Protein Interactions 294

9.4 Small-Molecule Allosteric Ligands 297

9.5 Quantitative Measurements of Enzyme Activation and Inhibition 302

9.6 Regulation of Protein Kinases 308

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

10.3 Effects of Inhibitors on Steady-State Parameters 324

10.4 Concentration-Response Plots of Enzyme Inhibition 333

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

10.8 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 K I 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

12.3 Distinguishing Between Modes of Inhibitor Interaction with Enzyme 382

12.4 Determining Reversibility 384

12.5 Examples of Slow-Binding Enzyme Inhibitors 386

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

13.3 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

14.3 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

16.4 Enzyme Families and Superfamilies 463

16.5 Enzyme Promiscuity as a Springboard of Evolution 467

16.6 Protein Dynamics and Conformational Selection in Evolution of Neofunctionality 474

16.7 Ancestral Enzyme Reconstruction 475

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

17.3 Enzyme Essentiality in Disease 492

17.4 Enzyme-Mediated Target Protein Degradation 524

17.5 The Role of Enzymology in Drug Discovery and Development 527

17.6 Summary 537

References and Further Reading 537

Index 543

Authors

Robert A. Copeland Accent Therapeutics, Inc.; Ki Consultant, LLC.