Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis

ISBN-13: 9781119793250 / Angielski / Twarda / 2023 / 500 str.

Copeland

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Preface to the Third Edition xviiPreface to the Second Edition xixPreface to the First Edition xxiAcknowledgments xxiii1 A Brief History of Enzymology 1Key Learning Points 11.1 Enzymes in Antiquity 21.2 Early Enzymology 31.3 The Development of Mechanistic Enzymology 41.4 Studies of Enzyme Structure 51.5 Enzymology Today 71.6 Summary 9References and Further Reading 92 Chemical Bonds and Reactions in Biochemistry 11Key Learning Points 112.1 Atomic and Molecular Orbitals 122.1.1 Atomic Orbitals 122.1.2 Molecular Orbitals 152.1.3 Hybrid Orbitals 162.1.4 Resonance and Aromaticity 182.1.5 Different Electronic Configurations Have Different Potential Energies 202.2 Thermodynamics of Chemical Reactions 222.2.1 The Transition State of Chemical Reactions 242.3 Acid-base Chemistry 272.4 Noncovalent Interactions in Reversible Binding 292.4.1 Electrostatic Interactions 302.4.2 Hydrogen Bonding 302.4.3 Hydrophobic Interactions 312.4.4 Van der Waals Forces 312.5 Rates of Chemical Reactions 332.5.1 Reaction Order 352.5.2 Reversible Chemical Reactions 362.5.3 Measurement of Initial Velocity 372.6 Summary 38References and Further Reading 383 Structural Components of Enzymes 39Key Learning Points 393.1 The Amino Acids 403.1.1 Properties of Amino-Acid Side Chains 423.1.1.1 Hydrophobicity 423.1.1.2 Hydrogen Bonding 423.1.1.3 Salt-Bridge Formation 433.1.2 Amino Acids as Acids and Bases 443.1.3 Cation and Metal Binding 453.1.4 Anion and Polyanion Binding 463.1.5 Covalent Bond Formation 463.1.5.1 Disulfide Bonds 463.1.5.2 Phosphorylation 463.1.5.3 Glycosylation 473.1.6 Steric Bulk 473.2 The Peptide Bond 483.3 Amino Acid Sequence or Primary Structure 513.4 Secondary Structure 543.4.1 The Right-Handed alpha Helix 553.4.2 The beta-Pleated Sheet 563.4.3 beta Turns 583.4.4 Other Secondary Structures 583.4.5 Supersecondary Structures 593.5 Tertiary Structure 603.5.1 Domains 623.6 Subunits and Quaternary Structure 643.7 Cofactors in Enzymes 673.8 Conformational Dynamics and Enzyme Function 703.9 Methods of Protein Structure Determination 753.9.1 X-ray Crystallography 763.9.2 NMR Spectroscopy 773.9.3 Cryo-Electron Microscopy (Cryo-EM) 783.10 Summary 79References and Further Reading 804 Protein-Ligand Binding Equilibria 83Key Learnings Points 834.1 The Equilibrium Dissociation Constant, Kd 844.2 The Kinetic Approach to Equilibrium 864.3 Binding Measurements at Equilibrium 884.3.1 Derivation of the Langmuir Isotherm 884.3.2 Multiple Binding Sites 914.3.2.1 Multiple Equivalent Binding Sites 914.3.2.2 Multiple Nonequivalent Binding Sites 924.3.2.3 Cooperative Interactions Among Multiple Binding Sites 924.3.3 Correction for Nonspecific Binding 934.4 Graphic Analysis of Equilibrium Ligand-Binding Data 944.4.1 Direct Plots on Semilog Scale 944.4.2 Linear Transformations of Binding Data: The Wolff Plots 974.5 Equilibrium Binding with Ligand Depletion (Tight Binding Interactions) 1004.6 Competition Among Ligands for a Common Binding Site 1014.7 Protein Dynamics in Receptor-Ligand Binding 1024.8 Orthosteric and Allosteric Ligand Binding Sites 1044.9 Experimental Methods for Measuring Ligand Binding 1054.9.1 Methods Based on Mass or Mobility Differences 1054.9.1.1 Equilibrium Dialysis 1054.9.1.2 Membrane Filtration Methods 1074.9.1.3 Size Exclusion Chromatography 1094.9.1.4 Microscale Thermophoresis 1114.9.2 Spectroscopic Methods 1134.9.2.1 Fluorescence Spectroscopy 1134.9.2.2 Surface Plasmon Resonance 1164.9.3 Ligand-Induced Protein Stabilization 1174.9.3.1 Thermal Denaturation of Proteins 1184.9.3.2 Chemical Denaturation of Proteins 1204.10 Summary 122References and Further Reading 1225 Steady-State Kinetics of Single-Substrate Enzyme Reactions 125Key Learning Points 1255.1 The Time Course of Enzymatic Reactions 1265.2 Effects of Substrate Concentration on Velocity 1275.3 The Rapid Equilibrium Model of Enzyme Kinetics 1295.4 The Steady-State Model of Enzyme Kinetics 1315.5 The Significance of kcat and Km 1345.5.1 Km 1355.5.2 kcat 1355.5.3 kcat/Km 1365.5.4 Diffusion-Controlled Reactions and Kinetic Perfection 1385.6 Experimental Measurement of kcat and Km 1395.6.1 Graphical Determinations from Untransformed Data 1395.6.2 Lineweaver-Burk Plots of Enzyme Kinetics 1425.7 Other Linear Transformations of Enzyme Kinetic Data 1475.7.1 Eadie-Hofstee Plots 1475.7.2 Hanes-Wolff Plots 1485.7.3 Eisenthal-Cornish-Bowden Direct Plots 1485.8 Measurements at Low Substrate Concentrations 1495.9 Deviations From Hyperbolic Kinetics 1505.10 Summary 153References and Further Reading 1536 Chemical Mechanisms in Enzyme Catalysis 155Key Learning Points 1556.1 Substrate-Active Site Complementarity 1566.2 Rate Enhancement Through Transition State Stabilization 1596.3 Chemical Mechanisms for Transition State Stabilization 1626.3.1 Approximation of Reactants 1636.3.2 Covalent Catalysis 1666.3.2.1 Nucleophilic Catalysis 1676.3.2.2 Electrophilic Catalysis 1686.3.3 General Acid/Base Catalysis 1706.3.4 Conformational Distortion 1756.3.5 Preorganized Active Site Complementarity to the Transition State 1806.4 The Serine Proteases: An Illustrative Example 1826.5 Enzymatic Reaction Nomenclature 1876.6 Summary 191References and Further Reading 1917 Experimental Measures of Steady-State Enzyme Activity 193Key Learning Points 1937.1 Initial Velocity Measurements 1947.1.1 Direct, Indirect, and Coupled Assays 1947.1.2 Analysis of Progress Curves: Measuring True Steady-State Velocity 2007.1.3 Continuous Versus End Point Assays 2037.1.4 Initiating, Mixing, and Stopping Reactions 2047.1.5 The Importance of Running Controls 2067.2 Detection Methods 2087.2.1 Assays Based on Optical Spectroscopy 2087.2.2 Absorption Measurements 2087.2.3 Choosing an Analytical Wavelength 2107.2.4 Optical Cells 2107.2.5 Errors in Absorption Spectroscopy 2127.2.6 Fluorescence Measurements 2137.2.7 Internal Fluorescence Quenching and Energy Transfer 2157.2.8 Errors in Fluorescence Measurements 2177.2.9 Radioisotopic Measurements 2207.2.10 Errors in Radioactivity Measurements 2237.2.11 Other Detection Methods 2237.3 Separation Methods in Enzyme Assays 2247.3.1 Separation of Proteins from Low Molecular Weight Solutes 2247.3.2 Chromatographic Separation Methods 2257.3.3 Electrophoretic Methods in Enzyme Assays 2307.4 Factors Affecting the Velocity of Enzymatic Reactions 2367.4.1 Enzyme Concentration 2377.4.2 pH Effects 2397.4.3 Temperature Effects 2457.4.4 Viscosity Effects 2477.4.5 Isotope Effects in Enzyme Kinetics 2497.5 Reporting Enzyme Activity Data 2527.6 Enzyme Stability 2537.6.1 Stabilizing Enzymes During Storage 2547.6.2 Enzyme Inactivation During Activity Assays 2557.7 Summary 258References and Further Reading 2588 Transient-State Kinetics 261Key Learning Points 2618.1 Timescale of Pre-Steady-State Turnover 2628.2 Instrumentation for Transient Kinetic Measurements 2648.3 Estimating Initial Conditions for Transient Kinetic Measurements 2668.4 Examples of Some Common Transient Kinetic Reaction Mechanisms 2678.4.1 One Step, Irreversible Binding 2678.4.2 One Step, Reversible Binding 2688.4.3 Consecutive, Irreversible Reaction 2688.4.4 Consecutive, Reversible Reaction with a Fast First Step (Pre-equilibrium Reaction) 2698.4.5 Consecutive, Reversible Reaction with a Fast Second Step (Enzyme Pre-isomerization) 2718.5 Examples of Transient Kinetic Studies from the Literature 2728.5.1 Study of Substrate and Inhibitor Interactions with the Alzheimer's Disease ß-Site Amyloid Precursor Protein-Cleaving Enzyme (BACE) 2728.5.2 Study of the Mechanism of Time-Dependent Inhibition of Staphylococcus aureusPolypeptide Deformylase 2758.6 Summary 277References and Further Reading 2789 Enzyme Regulation 279Key Learning Points 2799.1 Active and Inactive Conformational States 2809.2 Post-Translational Modifications 2819.2.1 Proteolytic Processing 2829.2.2 Covalent Modification of Amino Acid Side Chains 2889.3 Enzyme Regulation Through Protein-Protein Interactions 2949.4 Small-Molecule Allosteric Ligands 2979.4.1 Homotropic and Heterotropic Allostery 2989.4.2 Intramolecular and Intermolecular Allostery 2989.5 Quantitative Measurements of Enzyme Activation and Inhibition 3029.5.1 Thermodynamic Measurement of Activator-Enzyme Interactions 3039.5.2 Kinetic Measurement of Enzyme Activation by PTM 3069.6 Regulation of Protein Kinases 3089.6.1 Kinase Activation by PTM 3089.6.2 Kinase Regulation by Protein Association 3119.6.3 Kinase Activation by Oligomerization 3129.6.4 Kinase Regulation by Small-Molecule Binding 3139.6.5 Small-Molecule Mimicry of Intramolecular Allostery 3139.7 Summary 314References and Further Reading 31510 Reversible Inhibitors 317Key Learning Points 31710.1 Equilibrium Treatment of Reversible Inhibition 31910.2 Thermodynamic Modes of Reversible Inhibition 32110.2.1 Pure Competitive Inhibition, Exclusive Binding to Free Enzyme (E): alpha = infinity 32110.2.2 Mixed or Noncompetitive Inhibition 32210.2.2.1 Mixed Inhibitors That Bind Preferentially to the Free Enzyme (E): alpha >1 32210.2.2.2 Mixed Inhibitors That Bind Equipotently to E and ES: alpha=1 32210.2.2.3 Mixed Inhibitors That Bind Preferentially to the Enzyme-Substrate Complex (ES): alpha 1 32210.2.3 Pure Uncompetitive Inhibitors, Exclusive Binding to the Enzyme-Substrate Complex (ES): alpha<<1 32310.2.4 Partial Inhibitors 32310.3 Effects of Inhibitors on Steady-State Parameters 32410.3.1 Competitive Inhibitors 32510.3.2 Noncompetitive Inhibitors 32910.3.3 Uncompetitive Inhibitors 33010.3.4 Fitting of Untransformed Data 33210.4 Concentration-Response Plots of Enzyme Inhibition 33310.4.1 Concentration-Response Plots for Partial Inhibition 33610.5 Effects of Substrate Concentration on Inhibitor Concentration-Response Curves 33710.6 Mutually Exclusive Binding of Two Inhibitors 34010.7 Structure-Activity Relationships and Inhibitor Design 34310.7.1 SAR in the Absence of Enzyme Structural Information 34310.7.2 Inhibitor Design Based on Enzyme Structure 35010.8 Summary 353References and Further Reading 35411 Tight-Binding Inhibitors 357Key Learning Points 35711.1 Identifying Tight-Binding Inhibition 35811.2 Distinguishing Inhibitor Type for Tight-Binding Inhibitors 35911.3 Determining Ki for Tight-Binding Inhibitors 36211.4 Use of Tight-Binding Inhibitors to Determine Active Enzyme Concentration 36511.5 Summary 368 References and Further Reading 36812 Time-Dependent Inhibition 371Key Learning Points 37112.1 Progress Curves for Slow-Binding Inhibitors 37512.2 Distinguishing Between Slow-Binding Schemes 37812.2.1 Scheme B 37912.2.2 Scheme C 37912.2.3 Scheme D 38012.3 Distinguishing Between Modes of Inhibitor Interaction with Enzyme 38212.4 Determining Reversibility 38412.4.1 Enzyme-Inhibitor Residence Time 38512.5 Examples of Slow-Binding Enzyme Inhibitors 38612.5.1 Serine Proteases 38612.5.2 Prostaglandin G/H Synthase 38712.5.3 Chemical Modification as Probes of Enzyme Structure and Mechanism 39112.5.3.1 Amino Acid Selective Chemical Modification 39212.5.3.2 Substrate Protection Experiments 39412.5.3.3 Affinity Labels 39612.6 Summary 398References and Further Reading 39813 Enzyme Reactions with Multiple Substrates 401Key Learning Points 40113.1 Reaction Nomenclature 40213.2 Bi-Bi Reaction Mechanisms 40313.2.1 Random Ordered Bi-Bi Reactions 40313.2.2 Compulsory-Ordered Bi-Bi Reactions 40413.2.3 Double Displacement or Ping-Pong Bi-Bi Reactions 40613.3 Distinguishing Between Random and Compulsory-Ordered Mechanisms by Inhibition Pattern 40713.4 Isotope Exchange Studies for Distinguishing Reaction Mechanisms 40913.5 Using the King-Altman Method to Determine Velocity Equations 41113.6 Cleland's Net Rate Constant Method for Determining Vmax and Vmax/Km 41413.7 Summary 416References and Further Reading 41714 Enzyme-Macromolecule Interactions 419Key Learning Points 41914.1 Mutlitprotein Enzyme Complexes 42014.2 Enzyme Reactions on Macromolecular Substrates 42214.2.1 Differences Between Small Molecule and Protein Substrate Binding to Enzymes 42214.2.2 Factors Affecting Protein-Protein Interactions 42514.2.3 Separation of Binding and Catalytic Recognition Elements 42714.2.4 Noncompetitive Inhibition by Active Site Binding Molecules for Exosite Utilizing Enzymes 42914.2.5 Processive and Distributive Mechanisms of Catalysis 43014.2.6 Effect of Substrate Conformation on Enzyme Kinetics 43414.2.7 Inhibitor Binding to Substrates 43414.3 Summary 436References and Further Reading 43615 Cooperativity in Enzyme Catalysis 439Key Learning Points 43915.1 Historic Examples of Cooperativity and Allostery in Proteins 44115.2 Models of Allosteric Behavior 44515.3 Effects of Cooperativity on Velocity Curves 44915.4 Sigmoidal Kinetics for Nonallosteric Enzymes 45215.5 Summary 453References and Further Reading 45316 Evolution of Enzymes 455Key Learning Points 45516.1 Early Earth Conditions 45616.2 Natural Selection 45616.3 Genetic Alterations 45916.3.1 Single Nucleotide Polymorphisms (SNPs) 45916.3.2 Gene Duplication 46016.3.3 Deletions and Insertions 46116.3.4 Translocations and Inversions 46116.4 Enzyme Families and Superfamilies 46316.5 Enzyme Promiscuity as a Springboard of Evolution 46716.5.1 Evolution of Enzyme Steady State Parameters 47116.6 Protein Dynamics and Conformational Selection in Evolution of Neofunctionality 47416.7 Ancestral Enzyme Reconstruction 47516.7.1 Mechanism of Drug Selectivity for Gleevec 47716.7.2 Overcoming Epistasis to Define the Mechanism of Substrate Specificity 47816.7.3 Revealing Generalist to Specialist Evolution 47916.7.4 Ancestral Sequence Reconstruction as a Practical Tool 48016.8 Contemporary Enzyme Evolution 48016.9 Summary 483References and Further Reading 48317 Enzymes in Human Health 487Key Learning Points 48717.1 Enzymes as Therapeutic Agents 48717.2 Enzyme Inhibitors as Therapeutic Agents 48817.2.1 Properties of Small-Molecule Drugs 48917.2.2 Enzymes as Drug Targets 48917.3 Enzyme Essentiality in Disease 49217.3.1 Paralogues with Distinct Physiological Roles 49217.3.2 Distinct Orthologues in Infectious Diseases 49417.3.3 Diseases of Lifestyle, Environmental, and Aging 49717.3.4 Pathogenic Alterations to Enzyme Function 50117.3.4.1 Relating Genetic Alterations to Disease Essentiality 50217.3.4.2 Enzyme Overexpression 50517.3.4.3 Activating Mutations 50617.3.4.4 Chromosomal Translocations 51517.3.4.5 Synthetic Lethality 51817.3.5 Pro-Drug Activation by Enzymes 52217.4 Enzyme-Mediated Target Protein Degradation 52417.5 The Role of Enzymology in Drug Discovery and Development 52717.5.1 Enzyme Selectivity Assessment 52917.5.2 Correlating Enzyme Inhibition with Cellular Phenotype 53017.5.3 Hepatic Metabolism of Xenobiotics 53317.5.4 Mutation-Based Drug Resistance 53517.6 Summary 537References and Further Reading 537Index 543

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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.