• Preface
  • Purpose of This Book
  • Audience and Their Background
  • Organization
  • How to Use This Book
• Useful Constants and Conversion Factors
• Acknowledgments
• Contents
• About the Author
• Part I: Enzyme Catalysis - A Perspective
  • 1: Enzymes: Their Place in Biology
    • Suggested Reading
  • 2: Enzymes: Historical Aspects
    • 2.1 Biocatalysis: The Beginnings
    • 2.2 ``Enzyme´´: Conceptual Origin
    • 2.3 Key Developments in Enzymology
    • Reference
      • Suggested Reading
  • 3: Exploiting Enzymes: Technology and Applications
    • 3.1 Exploiting Natural Diversity
    • 3.2 Modifying Enzymes to Suit Requirements
    • 3.3 Genetic Engineering and Enzymes
    • 3.4 Summing Up
    • References
      • Suggested Reading
  • 4: On Enzyme Nomenclature and Classification
    • 4.1 What Is in the Name?
    • 4.2 Enzyme Diversity and Need for Systematics
    • 4.3 Enzyme Commission: Recommendations
    • 4.4 Some Concerns
    • References
  • 5: Hallmarks of an Enzyme Catalyst
    • 5.1 Catalysis
    • 5.2 Specificity
    • 5.3 Regulation
    • References
  • 6: Origins of Enzyme Catalytic Power
    • 6.1 Proximity and Orientation Effects
    • 6.2 Contribution by Electrostatics
    • 6.3 Metal Ions in Catalysis
    • 6.4 General Acid-Base Catalysis
    • 6.5 Covalent Catalysis
    • 6.6 Transition State Binding and Stabilization
    • References
      • Suggested Reading
  • 7: Which Enzyme Uses What Tricks?
    • References
  • 8: Structure and Catalysis: Conformational Flexibility and Protein Motion
    • References
      • Suggested Reading
• Part II: Enzyme Kinetic Practice and Measurements
  • 9: Chemical Kinetics: Fundamentals
    • 9.1 Measurement of Reaction Rates
    • 9.2 Factors that Influence Chemical Reaction Rates
    • 9.3 Reaction Progress and Its Concentration Dependence
    • 9.4 Temperature Dependence of Reaction Rates
    • 9.5 Catalysis
    • 9.6 Purpose of Kinetic Studies: Reaction Mechanism
    • Reference
      • Suggested Reading
  • 10: Concepts of Equilibrium and Steady State
    • 10.1 Chemical Reaction Equilibrium
    • 10.2 Binding Equilibrium
    • 10.3 Complex Reactions Involving Intermediates
    • References
      • Suggested Reading
  • 11: ES Complex and Pre-steady-state Kinetics
    • 11.1 ES Complex, Intermediates, and Transient Species
    • 11.2 Kinetic Competence of an Intermediate
    • 11.3 Pre-steady-state Kinetics
    • References
  • 12: Principles of Enzyme Assays
    • 12.1 Detection and Estimation Methods
    • 12.2 Enzyme Reaction Time Course
    • 12.3 Precautions and Practical Considerations
    • 12.4 Summing Up
    • References
      • Suggested Reading
  • 13: Good Kinetic Practices
    • 13.1 How to Assemble Enzyme Assay Mixtures
    • 13.2 pH and Ionic Strength Considerations
    • 13.3 Temperature Effects
    • 13.4 Summing Up
    • References
  • 14: Quantification of Catalysis and Measures of Enzyme Purity
    • 14.1 Enzyme Units, Specific Activity, and Turnover Number
    • 14.2 Enzyme Purification and Characterization
    • 14.3 Interpreting a Purification Table: Criteria of Enzyme Purity
    • 14.4 Unity of the Enzyme
    • 14.5 Summing Up
    • References
  • 15: Henri-Michaelis-Menten Equation
    • 15.1 Derivation of the Michaelis-Menten Equation
    • 15.2 Salient Features of Michaelis-Menten Equation
      • Calculating [S]0.9/[S]0.1
      • h and r: The Two Cooperativity Indices
    • 15.3 Significance of KM, Vmax, and kcat/KM
      • The Tradeoff Between kcat and KM
    • 15.4 Haldane Relationship: Equilibrium Constant Meets Kinetic Constants
      • Haldane Relationship and Isozymes
    • 15.5 Use and Misuse of Michaelis-Menten Equation
    • References
      • Suggested Reading
  • 16: More Complex Rate Expressions
    • 16.1 Investigating Enzyme Mechanisms Through Kinetics
      • Mechanism Building: The Process
    • 16.2 Notations and Nomenclature in Enzyme Kinetics
    • 16.3 Deriving Rate Equations for Complex Equilibria
      • 16.3.1 Algebraic Method
        • Rate Equation for the Equilibria Involving Two Enzyme Forms
      • 16.3.2 King-Altman Procedure
        • King-Altman Procedure for Equilibria with Two Enzyme Forms
      • 16.3.3 Net Rate Constant Method
        • Net Rate Constant Method for Linear Equilibria
      • 16.3.4 Other Methods
    • 16.4 Enzyme Kinetics and Common Sense
    • References
  • 17: Enzyme Kinetic Data: Collection and Analysis
    • 17.1 Obtaining Primary Data: Practical Aspects
      • 17.1.1 Reductionism in Experimental Design
      • 17.1.2 Choice of Substrate Concentrations
      • 17.1.3 Pilot Experiments and Iteration
      • 17.1.4 Importance of Measuring Initial Velocities
        • Monitoring NADP-Glutamate Dehydrogenase Reaction Progress
      • 17.1.5 Utility of the Integrated Form of Michaelis-Menten Equation
    • 17.2 Analyzing Data: The Basics
      • 17.2.1 Variation, Errors, and Statistics
    • 17.3 Plotting v Versus [S] Data
      • 17.3.1 The v Versus [S] Plot
      • 17.3.2 Direct Linear Plot
      • 17.3.3 v Versus log[S] Plot
      • 17.3.4 Hill Plot
    • 17.4 Linear Transforms of Michaelis-Menten Equation
      • 17.4.1 Lineweaver-Burk Plot
        • Practical Aspects of Double-Reciprocal Analysis
      • 17.4.2 Eadie-Hofstee Plot
      • 17.4.3 Woolf-Hanes Plot
    • 17.5 Summing Up
    • References
• Part III: Elucidation of Kinetic Mechanisms
  • 18: Approaches to Kinetic Mechanism: An Overview
    • 18.1 Which Study Gives What Kind of Information?
    • 18.2 Two Thumb Rules
  • 19: Analysis of Initial Velocity Patterns
    • 19.1 Intersecting Patterns
      • 19.1.1 Determination/Evaluation of Kinetic Constants and Replots
      • 19.1.2 Interpretation
    • 19.2 Parallel Patterns
      • 19.2.1 Determination/Evaluation of Kinetic Constants and Replots
      • 19.2.2 Interpretation
    • 19.3 Few Unique Variations
    • Appendix
    • References
  • 20: Enzyme Inhibition Analyses
    • 20.1 Reversible Versus Irreversible Inhibition
    • 20.2 Partial Versus Complete Inhibition
    • 20.3 Other Inhibitor Types
    • References
  • 21: Irreversible Inhibitions
    • 21.1 Chemical Modification Agents
    • 21.2 Affinity Labels
    • 21.3 Suicide Substrates
    • 21.4 Tight-Binding Inhibitors
    • References
  • 22: Reversible Inhibitions
    • 22.1 Competitive Inhibition
      • 22.1.1 Determination/Evaluation of Kinetic Constants and Replots
      • 22.1.2 Interpretation
    • 22.2 Uncompetitive Inhibition
      • 22.2.1 Determination/Evaluation of Kinetic Constants and Replots
      • 22.2.2 Interpretation
    • 22.3 Noncompetitive Inhibition
      • 22.3.1 Determination/Evaluation of Kinetic Constants and Replots
      • 22.3.2 Interpretation
    • 22.4 Reversible Inhibition Equilibria: Another Viewpoint
      • 22.4.1 Significance of α and β Values
    • 22.5 IC50 and Its Relation to KI of an Inhibitor
    • Appendix
    • References
  • 23: Alternate Substrate (Product) Interactions
    • 23.1 Substrate Inhibition
      • 23.1.1 Determination of Kinetic Constants and Their Significance
    • 23.2 Use of Alternate Substrates in Enzyme Studies
      • 23.2.1 Information About the Active Site Shape, Geometry, and Interactions
      • 23.2.2 Understanding Kinetic Mechanism
    • Reference
  • 24: pH Studies with Enzymes
    • 24.1 Enzyme pH Optimum
    • 24.2 pH Kinetic Profiles
    • 24.3 Identifying Groups Seen in pH Profiles
    • Reference
  • 25: Isotopes in Enzymology
    • 25.1 Enzyme Assays with a Radiolabeled Substrate
    • 25.2 Isotope Partitioning
    • References
  • 26: Isotope Exchanges at Equilibrium
    • 26.1 Partial Reactions and Ping-Pong Mechanism
    • 26.2 Sequential Mechanisms
    • References
  • 27: Isotope Effects in Enzymology
    • 27.1 Magnitude of the Observed Isotope Effect
    • 27.2 Experimental Approaches to Measure Isotope Effects
      • 27.2.1 Direct Comparison
      • 27.2.2 Equilibrium Perturbation
      • 27.2.3 Internal Competition Method
    • 27.3 Applications of KIEs in Enzymology:
      • 27.3.1 Elucidating Kinetic Mechanism
      • 27.3.2 Deciding Chemical Mechanism
      • 27.3.3 Understanding Enzyme Transition State
    • References
      • Suggested Reading
  • 28: From Kinetic Data to Mechanism and Back
    • 28.1 How to Relate Mechanisms with Steady-State Kinetic Data
      • 28.1.1 Ordered Mechanism
      • 28.1.2 Random Mechanism
      • 28.1.3 Ping-Pong Mechanism
    • 28.2 Assigning Kinetic Mechanisms: An Action Plan
    • 28.3 Practical Relevance of Enzyme Kinetics
      • 28.3.1 Affinity Chromatography and Protein Purification
      • 28.3.2 Dissection of Metabolism
      • 28.3.3 Enzyme-Targeted Drugs in Medicine
    • References
• Part IV: Chemical Mechanisms and Catalysis
  • 29: Chemical Reactivity and Molecular Interactions
    • 29.1 Atoms, Molecules, and Chemical Bonding
      • 29.1.1 Covalent Bonds
      • 29.1.2 Directional Property of Covalent Bonds
      • 29.1.3 Non-covalent Interactions and Intermolecular Forces
    • 29.2 Chemical Reaction Mechanisms
      • 29.2.1 Cleaving and Forming Covalent Bonds
      • 29.2.2 Logic of Pushing Electrons and Moving Bonds
        • Guidelines to a Chemical Mechanism
    • 29.3 Stereochemical Course of Reaction
    • 29.4 Common Organic Reaction Types
      • 29.4.1 Nucleophilic Displacements
      • 29.4.2 Elimination Reactions
      • 29.4.3 Carbon-Carbon Bond Formation
    • 29.5 Summing Up
    • Reference
      • Suggested Reading
  • 30: Acid-Base Chemistry and Catalysis
    • 30.1 Acids and Bases
      • Acid Dissociation Constant
    • 30.2 General Acid-Base Catalysis
      • Contributions of Specific and General Acid Catalysis
    • 30.3 Summing Up
    • References
  • 31: Nucleophilic Catalysis and Covalent Reaction Intermediates
    • 31.1 Nucleophiles and Electrophiles Available on the Enzyme
    • 31.2 Nucleophilic (Covalent) Catalysis
      • Criteria for Nucleophilic Catalysis
      • Catalysis by Nucleophile or Base?
    • 31.3 Covalent Reaction Intermediates
      • How Covalent Reaction Intermediates Are Formed?
    • 31.4 Detecting Intermediates and Establishing Their Catalytic Competence
    • 31.5 Summing Up
    • References
  • 32: Phosphoryl Group Chemistry and Importance of ATP
    • 32.1 Why Nature Chose Phosphates
    • 32.2 Chemical Mechanisms at the Phosphoryl Group
      • Phosphoryl Transfer Mechanism: Single or Double Displacement?
    • 32.3 Adenosine Triphosphate: Structure Relates to Function
    • 32.4 Investing Group Transfer Potential to Create Good Leaving Groups
    • 32.5 Summing Up
    • References
  • 33: Enzymatic Oxidation-Reduction Reactions
    • 33.1 What Are Oxidation-Reduction Reactions?
      • Redox Chemistry of Lactate Dehydrogenase Reaction
    • 33.2 How Enzymes Influence Redox Reaction Rates
    • 33.3 Mechanisms and Modes of Electron Transfer
    • 33.4 Pterine and Folate Cofactors
    • 33.5 Nicotinamide Cofactors
    • 33.6 Flavins and Flavoenzymes
    • 33.7 Reactions Involving Molecular Oxygen
    • 33.8 Summing Up
    • References
  • 34: Carboxylations and Decarboxylations
    • 34.1 Reactions and Reactivity of CO2
    • 34.2 Carboxylation Chemistry with Pyruvate and Phosphoenolpyruvate
      • Enzymes That Carboxylate PEP
    • 34.3 Cofactor-Assisted Carboxylations
      • Exchange Reactions Observed with Acetyl-CoA Carboxylase
    • 34.4 Decarboxylation Reactions
    • 34.5 Thiamine Pyrophosphate and α-Keto Acid Decarboxylations
      • Partial Reactions of Pyruvate Dehydrogenase Complex
    • 34.6 Summing Up
    • References
  • 35: Electrophilic Catalysis and Amino Acid Transformations
    • 35.1 Protein Electrophiles
    • 35.2 Reactions Involving Pyridoxal Phosphate (PLP)
    • 35.3 Summing Up
    • References
      • Suggested Reading
  • 36: Integrating Kinetic and Chemical Mechanisms: A Synthesis
    • 36.1 Competence of the Proposed Reaction Intermediate
    • 36.2 Glutamine Synthetase
    • 36.3 Glutamate Dehydrogenase
    • 36.4 Disaccharide Phosphorylases
    • 36.5 Acyl Transferases
    • 36.6 Chymotrypsin
    • 36.7 Aldolases and Transaldolase
    • 36.8 Ribonuclease A
    • 36.9 Interdependence of Kinetic and Chemical Mechanisms: A Summary
    • References
• Part V: Frontiers in Enzymology
  • 37: Regulation of Enzyme Activity
    • 37.1 Control of Enzyme Concentration
    • 37.2 Control of Enzyme Activity: Inhibition
    • 37.3 Control of Enzyme Activity: Cooperativity and Allostery
      • Oligomeric State, Subunit Cooperativity, and Metabolic Switch Behavior
    • 37.4 Isozymes and Regulation
    • 37.5 Covalent Modifications and Control
    • 37.6 Protein-Protein Interactions and Enzyme Control
    • 37.7 Compartmental Regulation and Membrane Transport
    • 37.8 Glutamine Synthetase: An Anthology of Control Mechanisms
    • 37.9 Summing Up
    • References
      • Suggested Reading
  • 38: In Vitro Versus In Vivo: Concepts and Consequences
    • 38.1 Why Michaelis-Menten Formalism Is Not Suitable In Vivo
    • 38.2 Concentration of Enzymes, Substrates, and Their Equilibria
    • 38.3 Avogadro´s Number Is a Very Big Number
    • 38.4 Diffusion, Crowding, and Enzyme Efficiency
    • 38.5 Consecutive Reactions and Metabolite Channeling
    • 38.6 Summing Up
    • References
  • 39: Future of Enzymology: An Appraisal
    • 39.1 Transition-State Analysis and Computational Enzymology
    • 39.2 Single-Molecule Enzymology
    • 39.3 Structure-Function Dissection of Enzyme Catalysis
    • 39.4 Designing Novel Catalysts
    • 39.5 Enzymes Made to Order
    • 39.6 Summing Up
    • References
      • General
      • Transition State Analysis and Computational Enzymology
      • Single Molecule Enzymology
      • Structure-Function Dissection of Enzyme Catalysis
      • Designing Novel Catalysts
      • Enzymes Made to Order
  • 40: Closure - Whither Enzymology
    • References
• Bibliography
  • Books
    • General and Historical
    • Enzyme Kinetics
    • Enzyme Chemical Mechanisms
    • Practical Enzymology
    • Enzymology Texts
    • Enzyme Regulation and Applications
  • Series
    • Volumes Covering Advances in Enzymology
  • Biochemistry Textbooks
    • For Background Material on Protein Structure, Metabolism and Gene Regulation

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