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

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 < 1
10.2.3 Pure Uncompetitive Inhibitors, Exclusive Binding to the Enzyme‐Substrate Complex (ES): α ≪ 1
10.2.4 Partial Inhibitors
10.3 EFFECTS OF INHIBITORS ON STEADY‐STATE PARAMETERS
10.3.1 Competitive Inhibitors
10.3.2 Noncompetitive Inhibitors
10.3.3 Uncompetitive Inhibitors
10.3.4 Fitting of Untransformed Data
10.4 CONCENTRATION‐RESPONSE PLOTS OF ENZYME INHIBITION
10.4.1 Concentration‐Response Plots for Partial Inhibition
10.5 EFFECTS OF SUBSTRATE CONCENTRATION ON INHIBITOR CONCENTRATION–RESPONSE CURVES
10.6 MUTUALLY EXCLUSIVE BINDING OF TWO INHIBITORS
10.7 STRUCTURE–ACTIVITY RELATIONSHIPS AND INHIBITOR DESIGN
10.7.1 SAR in the Absence of Enzyme Structural Information
10.7.2 Inhibitor Design Based on Enzyme Structure
10.8 SUMMARY
10.9 REFERENCES AND FURTHER READING
Chapter 11 TIGHT‐BINDING INHIBITORS
11.1 IDENTIFYING TIGHT‐BINDING INHIBITION
11.2 DISTINGUISHING INHIBITOR TYPE FOR TIGHT‐BINDING INHIBITORS
11.3 DETERMINING Ki FOR TIGHT‐BINDING INHIBITORS
11.4 USE OF TIGHT‐BINDING INHIBITORS TO DETERMINE ACTIVE ENZYME CONCENTRATION
11.5 SUMMARY
11.6 REFERENCES AND FURTHER READING
Chapter 12 TIME‐DEPENDENT INHIBITION
12.1 PROGRESS CURVES FOR SLOW‐BINDING INHIBITORS
12.2 DISTINGUISHING BETWEEN SLOW‐BINDING SCHEMES
12.2.1 Scheme B
12.2.2 Scheme C
12.2.3 Scheme D
12.3 DISTINGUISHING BETWEEN MODES OF INHIBITOR INTERACTION WITH ENZYME
12.4 DETERMINING REVERSIBILITY
12.4.1 Enzyme‐Inhibitor Residence Time
12.5 EXAMPLES OF SLOW‐BINDING ENZYME INHIBITORS
12.5.1 Serine Proteases
12.5.2 Prostaglandin G/H Synthase
12.5.3 Chemical Modification as Probes of Enzyme Structure and Mechanism
12.5.3.1 Amino Acid Selective Chemical Modification
12.5.3.2 Substrate Protection Experiments
12.5.3.3 Affinity Labels
12.6 SUMMARY
12.7 REFERENCES AND FURTHER READING
Chapter 13 ENZYME REACTIONS WITH MULTIPLE SUBSTRATES
13.1 REACTION NOMENCLATURE
13.2 Bi–Bi REACTION MECHANISMS
13.2.1 Random Ordered Bi–Bi Reactions
13.2.2 Compulsory‐Ordered Bi–Bi Reactions
13.2.3 Double Displacement or Ping–Pong Bi–Bi Reactions
13.3 DISTINGUISHING BETWEEN RANDOM AND COMPULSORY‐ORDERED MECHANISMS BY INHIBITION PATTERN
13.4 ISOTOPE EXCHANGE STUDIES FOR DISTINGUISHING REACTION MECHANISMS
13.5 USING THE KING–ALTMAN METHOD TO DETERMINE VELOCITY EQUATIONS
13.6 CLELAND'S NET RATE CONSTANT METHOD FOR DETERMINING VMAX AND VMAX/Km
13.7 SUMMARY
13.8 REFERENCES AND FURTHER READING
Chapter 14 Enzyme–Macromolecule Interactions
14.1 Mutlitprotein Enzyme Complexes
14.2 Enzyme Reactions on Macromolecular Substrates
14.2.1 Differences Between Small Molecule and Protein Substrate Binding to Enzymes
14.2.2 Factors Affecting Protein–Protein Interactions
14.2.3 Separation of Binding and Catalytic Recognition Elements
14.2.4 Noncompetitive Inhibition by Active Site Binding Molecules for Exosite Utilizing Enzymes
14.2.5 Processive and Distributive Mechanisms of Catalysis
14.2.6 Effect of Substrate Conformation on Enzyme Kinetics
14.2.7 Inhibitor Binding to Substrates
14.3 Summary
14.4 References and Further Reading
Chapter 15 Cooperativity in Enzyme Catalysis
15.1 Historic Examples of Cooperativity and Allostery in Proteins
15.2 Models of Allosteric Behavior
15.3 Effects of Cooperativity on Velocity Curves
15.4 Sigmoidal Kinetics for Nonallosteric Enzymes
15.5 Summary
15.6 References and Further Reading
Chapter 16 EVOLUTION OF ENZYMES
16.1 EARLY EARTH CONDITIONS
16.2 NATURAL SELECTION
16.3 GENETIC ALTERATIONS
16.3.1 Single Nucleotide Polymorphisms (SNPs)
16.3.2 Gene Duplication
16.3.3 Deletions and Insertions
16.3.4 Translocations and Inversions
16.4 ENZYME FAMILIES AND SUPERFAMILIES
16.5 ENZYME PROMISCUITY AS A SPRINGBOARD OF EVOLUTION
16.5.1 Evolution of Enzyme Steady State Parameters
16.6 PROTEIN DYNAMICS AND CONFORMATIONAL SELECTION IN EVOLUTION OF NEOFUNCTIONALITY
16.7 ANCESTRAL ENZYME RECONSTRUCTION
16.7.1 Mechanism of Drug Selectivity for Gleevec
16.7.2 Overcoming Epistasis to Define the Mechanism of Substrate Specificity
16.7.3 Revealing Generalist to Specialist Evolution
16.7.4 Ancestral Sequence Reconstruction as a Practical Tool
16.8 CONTEMPORARY ENZYME EVOLUTION
16.9 SUMMARY
16.10 REFERENCES AND FURTHER READING
Chapter 17 ENZYMES IN HUMAN HEALTH
17.1 ENZYMES AS THERAPEUTIC AGENTS
17.2 ENZYME INHIBITORS AS THERAPEUTIC AGENTS
17.2.1 Properties of Small‐Molecule Drugs
17.2.2 Enzymes as Drug Targets
17.3 ENZYME ESSENTIALITY IN DISEASE
17.3.1 Paralogues with Distinct Physiological Roles
17.3.2 Distinct Orthologues in Infectious Diseases
17.3.3 Diseases of Lifestyle, Environmental, and Aging
17.3.4 Pathogenic Alterations to Enzyme Function
17.3.4.1 Relating Genetic Alterations to Disease Essentiality
17.3.4.2 Enzyme Overexpression
17.3.4.3 Activating Mutations
17.3.4.4 Chromosomal Translocations
17.3.4.5 Synthetic Lethality
17.3.5 Pro‐Drug Activation by Enzymes
17.4 ENZYME‐MEDIATED TARGET PROTEIN DEGRADATION
17.5 THE ROLE OF ENZYMOLOGY IN DRUG DISCOVERY AND DEVELOPMENT
17.5.1 Enzyme Selectivity Assessment
17.5.2 Correlating Enzyme Inhibition with Cellular Phenotype
17.5.3 Hepatic Metabolism of Xenobiotics
17.5.4 Mutation‐Based Drug Resistance
17.6 SUMMARY
17.7 REFERENCES AND FURTHER READING
INDEX
EULA