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

Description

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 moreNew chapters on enzyme regulation, enzyme-macromolecule interactions, enzyme evolution, and enzymes in human healthKey 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. Cover Title Page Copyright Contents PREFACE TO THE THIRD EDITION PREFACE TO THE SECONDEDITION PREFACE TO THE FIRST EDITION ACKNOWLEDGMENTS Chapter 1 A BRIEF HISTORY OF ENZYMOLOGY 1.1 ENZYMES IN ANTIQUITY 1.2 EARLY ENZYMOLOGY 1.3 THE DEVELOPMENT OF MECHANISTIC ENZYMOLOGY 1.4 STUDIES OF ENZYME STRUCTURE 1.5 ENZYMOLOGY TODAY 1.6 SUMMARY 1.7 REFERENCES AND FURTHER READING Chapter 2 Chemical Bonds and Reactions in Biochemistry 2.1 Atomic and Molecular Orbitals 2.1.1 Atomic Orbitals 2.1.2 Molecular Orbitals 2.1.3 Hybrid Orbitals 2.1.4 Resonance and Aromaticity 2.1.5 Different Electronic Configurations Have Different Potential Energies 2.2 Thermodynamics of Chemical Reactions 2.2.1 The Transition State of Chemical Reactions 2.3 Acid–base Chemistry 2.4 Noncovalent Interactions in Reversible Binding 2.4.1 Electrostatic Interactions 2.4.2 Hydrogen Bonding 2.4.3 Hydrophobic Interactions 2.4.4 Van der Waals Forces 2.5 Rates of Chemical Reactions 2.5.1 Reaction Order 2.5.2 Reversible Chemical Reactions 2.5.3 Measurement of Initial Velocity 2.6 Summary 2.7 References and Further Reading Chapter 3 Structural Components of Enzymes 3.1 THE AMINO ACIDS 3.1.1 Properties of Amino‐Acid Side Chains 3.1.1.1 Hydrophobicity 3.1.1.2 Hydrogen Bonding 3.1.1.3 Salt‐Bridge Formation 3.1.2 Amino Acids as Acids and Bases 3.1.3 Cation and Metal Binding 3.1.4 Anion and Polyanion Binding 3.1.5 Covalent Bond Formation 3.1.5.1 Disulfide Bonds 3.1.5.2 Phosphorylation 3.1.5.3 Glycosylation 3.1.6 Steric Bulk 3.2 THE PEPTIDE BOND 3.3 AMINO ACID SEQUENCE OR PRIMARY STRUCTURE 3.4 SECONDARY STRUCTURE 3.4.1 The Right‐Handed &bfitalpha; Helix 3.4.2 The &bfitbeta;‐Pleated Sheet 3.4.3 &bfitbeta; Turns 3.4.4 Other Secondary Structures 3.4.5 Supersecondary Structures 3.5 TERTIARY STRUCTURE 3.5.1 Domains 3.6 SUBUNITS AND QUATERNARY STRUCTURE 3.7 COFACTORS IN ENZYMES 3.8 CONFORMATIONAL DYNAMICS AND ENZYME FUNCTION 3.9 METHODS OF PROTEIN STRUCTURE DETERMINATION 3.9.1 X‐ray Crystallography 3.9.2 NMR Spectroscopy 3.9.3 Cryo‐Electron Microscopy (Cryo‐EM) 3.10 SUMMARY 3.11 REFERENCES AND FURTHER READING Chapter 4 PROTEIN–LIGAND BINDING EQUILIBRIA 4.1 THE EQUILIBRIUM DISSOCIATION CONSTANT, K 4.2 THE KINETIC APPROACH TO EQUILIBRIUM 4.3 BINDING MEASUREMENTS AT EQUILIBRIUM 4.3.1 Derivation of the Langmuir Isotherm 4.3.2 Multiple Binding Sites 4.3.2.1 Multiple Equivalent Binding Sites 4.3.2.2 Multiple Nonequivalent Binding Sites 4.3.2.3 Cooperative Interactions Among Multiple Binding Sites 4.3.3 Correction for Nonspecific Binding 4.4 GRAPHIC ANALYSIS OF EQUILIBRIUM LIGAND‐BINDING DATA 4.4.1 Direct Plots on Semilog Scale 4.4.2 Linear Transformations of Binding Data: The Wolff Plots 4.5 EQUILIBRIUM BINDING WITH LIGAND DEPLETION (TIGHT BINDING INTERACTIONS) 4.6 COMPETITION AMONG LIGANDS FOR A COMMON BINDING SITE 4.7 PROTEIN DYNAMICS IN RECEPTOR–LIGAND BINDING 4.8 ORTHOSTERIC AND ALLOSTERIC LIGAND BINDING SITES 4.9 EXPERIMENTAL METHODS FOR MEASURING LIGAND BINDING 4.9.1 Methods Based on Mass or Mobility Differences 4.9.1.1 Equilibrium Dialysis 4.9.1.2 Membrane Filtration Methods 4.9.1.3 Size Exclusion Chromatography 4.9.1.4 Microscale Thermophoresis 4.9.2 Spectroscopic Methods 4.9.2.1 Fluorescence Spectroscopy 4.9.2.2 Surface Plasmon Resonance 4.9.3 Ligand‐Induced Protein Stabilization 4.9.3.1 Thermal Denaturation of Proteins 4.9.3.2 Chemical Denaturation of Proteins 4.10 SUMMARY 4.11 REFERENCES AND FURTHER READING Chapter 5 STEADY‐STATE KINETICS OF SINGLE‐SUBSTRATE ENZYME REACTIONS 5.1 THE TIME COURSE OF ENZYMATIC REACTIONS 5.2 EFFECTS OF SUBSTRATE CONCENTRATION ON VELOCITY 5.3 THE RAPID EQUILIBRIUM MODEL OF ENZYME KINETICS 5.4 THE STEADY‐STATE MODEL OF ENZYME KINETICS 5.5 THE SIGNIFICANCE OF AND K 5.5.1 Km 5.5.2 kcat 5.5.3 kcat/Km 5.5.4 Diffusion‐Controlled Reactions and Kinetic Perfection 5.6 EXPERIMENTAL MEASUREMENT OF AND K 5.6.1 Graphical Determinations from Untransformed Data 5.6.2 Lineweaver–Burk Plots of Enzyme Kinetics 5.7 OTHER LINEAR TRANSFORMATIONS OF ENZYME KINETIC DATA 5.7.1 Eadie–Hofstee Plots 5.7.2 Hanes–Wolff Plots 5.7.3 Eisenthal–Cornish‐Bowden Direct Plots 5.8 MEASUREMENTS AT LOW SUBSTRATE CONCENTRATIONS 5.9 DEVIATIONS FROM HYPERBOLIC KINETICS 5.10 SUMMARY 5.11 REFERENCES AND FURTHER READING Chapter 6 CHEMICAL MECHANISMS IN ENZYME CATALYSIS 6.1 Substrate–Active Site Complementarity 6.2 RATE ENHANCEMENT THROUGH TRANSITION STATE STABILIZATION 6.3 CHEMICAL MECHANISMS FOR TRANSITION STATE STABILIZATION 6.3.1 Approximation of Reactants 6.3.2 Covalent Catalysis 6.3.2.1 Nucleophilic Catalysis 6.3.2.2 Electrophilic Catalysis 6.3.3 General Acid/Base Catalysis 6.3.4 Conformational Distortion 6.3.5 Preorganized Active Site Complementarity to the Transition State 6.4 THE SERINE PROTEASES: AN ILLUSTRATIVE EXAMPLE 6.5 ENZYMATIC REACTION NOMENCLATURE 6.6 SUMMARY 6.7 REFERENCES AND FURTHER READING Chapter 7 EXPERIMENTAL MEASURES OF STEADY‐STATE ENZYME ACTIVITY 7.1 INITIAL VELOCITY MEASUREMENTS 7.1.1 Direct, Indirect, and Coupled Assays 7.1.2 Analysis of Progress Curves: Measuring True Steady‐State Velocity 7.1.3 Continuous Versus End Point Assays 7.1.4 Initiating, Mixing, and Stopping Reactions 7.1.5 The Importance of Running Controls 7.2 DETECTION METHODS 7.2.1 Assays Based on Optical Spectroscopy 7.2.2 Absorption Measurements 7.2.3 Choosing an Analytical Wavelength 7.2.4 Optical Cells 7.2.5 Errors in Absorption Spectroscopy 7.2.6 Fluorescence Measurements 7.2.7 Internal Fluorescence Quenching and Energy Transfer 7.2.8 Errors in Fluorescence Measurements 7.2.9 Radioisotopic Measurements 7.2.10 Errors in Radioactivity Measurements 7.2.11 Other Detection Methods 7.3 SEPARATION METHODS IN ENZYME ASSAYS 7.3.1 Separation of Proteins from Low Molecular Weight Solutes 7.3.2 Chromatographic Separation Methods 7.3.3 Electrophoretic Methods in Enzyme Assays 7.4 FACTORS AFFECTING THE VELOCITY OF ENZYMATIC REACTIONS 7.4.1 Enzyme Concentration 7.4.2 pH Effects 7.4.3 Temperature Effects 7.4.4 Viscosity Effects 7.4.5 Isotope Effects in Enzyme Kinetics 7.5 REPORTING ENZYME ACTIVITY DATA 7.6 ENZYME STABILITY 7.6.1 Stabilizing Enzymes During Storage 7.6.2 Enzyme Inactivation During Activity Assays 7.7 SUMMARY 7.8 REFERENCES AND FURTHER READING Chapter 8 TRANSIENT‐STATE KINETICS 8.1 TIMESCALE OF PRE‐STEADY‐STATE TURNOVER 8.2 INSTRUMENTATION FOR TRANSIENT KINETIC MEASUREMENTS 8.3 ESTIMATING INITIAL CONDITIONS FOR TRANSIENT KINETIC MEASUREMENTS 8.4 EXAMPLES OF SOME COMMON TRANSIENT KINETIC REACTION MECHANISMS 8.4.1 One Step, Irreversible Binding 8.4.2 One Step, Reversible Binding 8.4.3 Consecutive, Irreversible Reaction 8.4.4 Consecutive, Reversible Reaction with a Fast First Step (Pre‐equilibrium Reaction) 8.4.5 Consecutive, Reversible Reaction with a Fast Second Step (Enzyme Pre‐isomerization) 8.5 EXAMPLES OF TRANSIENT KINETIC STUDIES FROM THE LITERATURE 8.5.1 Study of Substrate and Inhibitor Interactions with the Alzheimer's Disease &rmbeta;‐Site Amyloid Precursor Protein‐Cleaving Enzyme (BACE) 8.5.2 Study of the Mechanism of Time‐Dependent Inhibition of Staphylococcus aureus Polypeptide Deformylase 8.6 SUMMARY 8.7 REFERENCES AND FURTHER READING Chapter 9 ENZYME REGULATION 9.1 Active and Inactive Conformational States 9.2 Post‐Translational Modifications 9.2.1 Proteolytic Processing 9.2.2 Covalent Modification of Amino Acid Side Chains 9.3 Enzyme Regulation Through Protein–Protein Interactions 9.4 Small‐Molecule Allosteric Ligands 9.4.1 Homotropic and Heterotropic Allostery 9.4.2 Intramolecular and Intermolecular Allostery 9.5 Quantitative Measurements of Enzyme Activation and Inhibition 9.5.1 Thermodynamic Measurement of Activator–Enzyme Interactions 9.5.2 Kinetic Measurement of Enzyme Activation by PTM 9.6 Regulation of Protein Kinases 9.6.1 Kinase Activation by PTM 9.6.2 Kinase Regulation by Protein Association 9.6.3 Kinase Activation by Oligomerization 9.6.4 Kinase Regulation by Small‐Molecule Binding 9.6.5 Small‐Molecule Mimicry of Intramolecular Allostery 9.7 Summary 9.8 References and Further Reading Chapter 10 REVERSIBLE INHIBITORS 10.1 EQUILIBRIUM TREATMENT OF REVERSIBLE INHIBITION 10.2 THERMODYNAMIC MODES OF REVERSIBLE INHIBITION 10.2.1 Pure Competitive Inhibition, Exclusive Binding to Free Enzyme (E): α = ∞ 10.2.2 Mixed or Noncompetitive Inhibition 10.2.2.1 Mixed Inhibitors That Bind Preferentially to the Free Enzyme (E): α > 1 10.2.2.2 Mixed Inhibitors That Bind Equipotently to E and ES: α = 1 10.2.2.3 Mixed Inhibitors That Bind Preferentially to the Enzyme–Substrate Complex (ES): α