Enzymes

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