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 Helix 55 3.4.2 The -Pleated Sheet 56 3.4.3 Turns 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 K m 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 Ac.