Laboratory Guide to Enzymology 1st Edition by Geoffrey Holdgate, Antonia Turberville, Alice Lanne ISBN 978-1394179794 1394179790

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ISBN 10:  1394179790

ISBN 13: 978-1394179794

Author: Geoffrey A. Holdgate, Antonia Turberville, Alice Lanne 

LABORATORY GUIDE TO ENZYMOLOGY
An accessible guide to understanding the foundations of enzymology at its application in drug discovery

Enzymes are highly specialized proteins necessary for performing specific biochemical reactions essential for life in all organisms. In disease, the functioning of these enzymes can become altered and, therefore, enzymes represent a large class of key targets for drug discovery. In order to successfully target dysfunctional enzymes pharmaceutically, the unique mechanism of each enzyme must be understood through thorough and in-depth kinetic analysis. The topic of enzymology can appear challenging due its interdisciplinary nature combining concepts from biology, chemistry, and mathematics.

Laboratory Guide to Enzymology brings together the theory of enzymology and associated lab-based work to offer a practical, accessible guide encompassing all three scientific disciplines. Beginning with a brief introduction to proteins and enzymes, the book slowly immerses the reader into the foundations of enzymology and how it can be used in drug discovery using modern methods of experimentation. The result is a detailed but highly readable volume detailing the basis of drug discovery research.

Laboratory Guide to Enzymology readers will also find:

Descriptions of key concepts in enzymology
Examples of drugs targeting different enzymes via different mechanisms
Detailed discussion about many areas of enzymology such as binding and steady-state kinetics, assay development, and enzyme inhibition and activation
Laboratory Guide to Enzymology is ideal for all pharmaceutical and biomedical researchers working in enzymology and assay development, as well as advanced students in the biochemical or biomedical sciences looking to develop a working knowledge of this field of research.

Table of contents: 

Chapter 1: Introduction to Proteins and Enzymes

• Protein Structure

  • Primary Structure

  • Secondary Structure

    • The Alpha Helix

    • The Beta Sheet

    • Loops

  • Tertiary Structure

    • Domains, Folds, and Motifs

  • Quaternary Structure

  • Protein Structure Prediction

• Enzymes

  • Properties of Enzymes

    • Catalysis

    • Specificity

    • Regulation

    • Stability

• References

Chapter 2: Binding Equilibria and Kinetics

• Introduction to Chemical Kinetics

  • Zero-order Reactions

  • First-order Reactions

  • Second-order Reactions

  • Pseudo-First-Order Reactions

  • Temperature Dependence of Rate Constants

• Introduction to Binding Kinetics

• Ligand Binding to Single Binding Site

  • Specific Versus Non-specific Binding

• Kinetic Approach to Equilibrium

• Methods for Measuring Protein-ligand Binding

• Ligand Depletion (Tight Binding)

• Ligand Binding to Multiple Binding Sites

  • Identical Independent Binding Sites

  • Non-identical Independent Binding Sites

  • Cooperativity

• Ligand Competition

• References

Chapter 3: Protein Quality Control and Handling

• Introduction

• Confirming Protein Identity

  • Intact Mass Measurement by Mass Spectrometry (MS)

  • Peptide Mapping

  • Edman Sequencing

• Protein Purity

  • SDS-PAGE

  • Dynamic Light Scattering (DLS)

  • Analytical Gel Filtration

  • Analytical Ultracentrifugation

• Concentration

  • UV-Vis Spectrum

  • Bradford Assay

• Functionality

  • Ligand Binding

    • Isothermal Titration Calorimetry (ITC)

    • Surface Plasmon Resonance (SPR)

  • Functional Studies

• Stability

  • Differential Scanning Calorimetry (DSC)

  • Differential Scanning Fluorimetry (DSF)

  • Circular Dichroism

  • Selwyn’s Test

• References

Chapter 4: Buffers and Their Use in the Study of Enzyme Mechanism

• Introduction

  • Ionization and pKa

  • pH

  • Henderson-Hasselbalch Equation

  • Buffers

• Buffering Capacity

• Ionic Strength

• Change in pH with Temperature

• Choice of Buffer

• Characteristics of Ionizing Groups in Proteins

• Effect of pH on Enzyme Activity

  • Assumptions Required for the Analysis of pH Dependence

  • General Rate Equation for pH Dependence

  • Fitting pH Dependence

  • Double Ionizing Systems

  • Singularly Ionizing Systems

• Effect of Solvent and Ionic Strength

• References

Chapter 5: Steady-State Assays and Their Design

• Introduction

• The Pre-steady State

• Steady-State Assays

• Assay Development

  • Requirements for Method Development

  • Different Types of Enzyme Assays

    • Direct Continuous Assay

    • Indirect Assays

      • Discontinuous Indirect Assays

      • Continuous Indirect Assays

      • Coupled Assays

• Blank Rates

• The Assay Development Process

  • Initial Assay Scoping

  • Substrate Dependence

    • Non-Michaelian Kinetics

    • Multiple Substrates

  • Plate Type

• Assay Optimization

  • Factorial Experimental Design (FED)

  • Coupling Enzyme Considerations

• Kinetic Characterization

  • Substrate Concentration

• Assay Adaptability

  • Tool Compounds

  • DMSO Tolerance

  • Assay Stability

  • Triage Assays

• Assay Evaluation (Validation)

  • Calculations

  • Assessing Plate Uniformity

  • Acceptable Assay Performance Criteria

• References

Chapter 6: Enzyme Inhibition

• Introduction

• Substrate and Product Inhibition

• Reversibility

  • Testing for Irreversible Inhibition

  • Rapidly Reversible Inhibition

• The IC50 Value

  • Determining the IC50 Value

  • Use of pIC50

  • Comparison of Potency

  • Concentration-response Curve Analysis

    • Bell-shaped Behavior

    • Weakly Active Compounds

    • Steep Curves

    • Partial Curves

    • Noisy Data

• Identity of Substrate

• Effect of Enzyme Concentration – Tight-binding Inhibition

• Slow-binding Inhibition

  • Progress Curves for Slow-binding Inhibition

• Slow, Tight-binding Inhibition

  • Conditions where Detection of Slow-binding may be Precluded

• Irreversible Inhibition

• Presence of Two Inhibitors

• Non-specific Inhibition

  • Common Technology Hitters

    • UV-light Interference

    • Detection System Interference

  • Chemical Reactivity

  • Aggregators

  • Redox Reactivity

    • Oxidation of Aromatic Amino Acid Residues

    • Protein Unfolding

  • Denaturation

  • Metal Ion Contamination

• References

Chapter 7: Enzyme Activation and Its Comparison with Inhibition

• Introduction

• Mechanisms for Enzyme Activation

  • Essential Activation

    • Essential Cationic Activation

  • Non-essential Activation

    • Non-essential Cationic Activation

  • A Comment on Nomenclature: K-type and V-type Classification

  • De-inhibition

• Challenges for Identifying Non-essential Enzyme Activators

  • Enzymes have Evolved to be Active

  • Lack of Tool Compounds

  • Maintaining Steady State

  • Mechanistic Considerations

  • Assay Design and Variability

• Addressing the Challenges of Activator Discovery

• References

Chapter 8: Mechanism of Action

• Introduction

• Mechanisms of Inhibition

  • Competitive Inhibition

  • Mixed Noncompetitive and Pure Noncompetitive Inhibition

  • Uncompetitive Inhibition

• Choosing Between Different Types of Inhibition

• Interpretation of Mechanism of Inhibition

• Effect of Multiple Substrates and Assignment of Mechanism

• Binding Site may not Equal Mechanism

  • Substrate Competitive Inhibitors at Allosteric Sites

  • Noncompetitive Binding giving Competitive Inhibition

  • Competitive Binding giving Uncompetitive Inhibition

• Specificity

  • Effect of Increasing [S]

  • Effect of [I]

• Activation Mechanisms and Comparison with Inhibition

• References

Chapter 9: Data Analysis

• Introduction

• Statistical Analysis of Enzyme Kinetic Data

• Least Squares Fitting

• Nonlinear Regression

• Weighting of Experimental Data

• Evaluation of Potential Different Models

  • Distribution of Residuals

    • The Runs Test

  • Magnitude of Standard Errors

  • Quantitative Evaluation

    • F-test

    • Akaike Information Criterion

    • Absolute Goodness of Fit

• Least Significant Ratio

• Two Independent Variables

  • Global Fitting

  • Reducing and Repeating and Reducing Only

    • Reducing Only

    • Reducing and Repeating

• References

Chapter 10: Molecular Interactions Between Proteins and Small Molecules

• Introduction

• Binding Affinity is a Function of Difference Energy

• Interactions Between Charged or Polar Groups

  • Electrostatic Interactions

  • Hydrogen Bonds

• Nonpolar Interactions

  • van der Waals Interactions

  • Hydrophobic Interactions

• Changes in Hydrophobicity on Chemical Substitution

• Entropy

• References

Chapter 11: Applications in Drug Discovery

• Introduction

• Pre-screening

  • Enzyme Considerations

  • Substrate Considerations

  • Enzyme Mechanism Considerations

• Post-screening

  • Measuring Potency

  • Reversibility

  • Inhibitor Mechanistic Characterization

    • Combining Enzyme Kinetics and Biophysics

  • Tight Binding

  • Undesired Mechanisms

• In Vitro Versus In Cellulo Enzyme Kinetics

• References

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Tags: Geoffrey Holdgate, Antonia Turberville, Alice Lanne, Laboratory Guide