Geoffrey A. Holdgate, Antonia Turberville, Alice Lanne

Laboratory Guide to Enzymology

• Broschiertes Buch

Produktbeschreibung

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 andenzymes, 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.

Produktdetails

• Verlag: Wiley / Wiley & Sons
• Artikelnr. des Verlages: 1W394179790
• 1. Auflage
• Seitenzahl: 304
• Erscheinungstermin: 1. März 2024
• Englisch
• Abmessung: 255mm x 177mm x 18mm
• Gewicht: 579g
• ISBN-13: 9781394179794
• ISBN-10: 1394179790
• Artikelnr.: 68763510

Autorenporträt

Geoffrey A. Holdgate is Senior Principal Scientist in Discovery Sciences for BioPharmaceuticals Research and Development at AstraZeneca. Antonia Turberville, PhD, is a Senior Scientist in Discovery Sciences for Biopharmaceuticals R&D at AstraZeneca. Alice Lanne, PhD, is a Senior Scientist in Discovery Sciences for BioPharmaceuticals R&D at AstraZeneca.

Inhaltsangabe

Preface xiii

Acknowledgments xv

1 Introduction to Proteins and Enzymes 1

1.1 Protein Structure 1

1.1.1 Primary Structure 1

1.1.2 Secondary Structure 1

1.1.3 Tertiary Structure 5

1.1.4 Quaternary Structure 6

1.1.5 Protein Structure Prediction 6

1.2 Enzymes 9

References 15

2 Binding Equilibria and Kinetics 17

2.1 Introduction to Chemical Kinetics 17

2.2 Introduction to Binding Kinetics 22

2.3 Ligand Binding to Single Binding Site 23

2.4 Kinetic Approach to Equilibrium 26

2.5 Methods for Measuring Protein-ligand Binding 27

2.6 Ligand Depletion (Tight Binding) 29

2.8 Ligand Competition 34

References 35

3 Protein QC and Handling 37

3.1 Introduction 37

3.2 Confirming Protein Identity 37

3.2.2 Peptide Mapping 38

3.2.3 Edman Sequencing 39

3.3 Protein Purity 39

3.3.1 Sds-page 40

3.3.2 Dynamic Light Scattering (DLS) 40

3.3.3 Analytical Gel Filtration 41

3.3.4 Analytical Ultracentrifugation 42

3.4 Concentration 43

3.4.1 UV-Vis Spectrum 44

3.4.2 Bradford Assay 44

3.5 Functionality 46

3.5.1 Ligand Binding 46

3.5.1.1 Itc 46

3.5.1.2 Spr 46

3.5.2 Functional Studies 46

3.6 Stability 48

3.6.1 Differential Scanning Calorimetry (DSC) 48

3.6.2 Differential Scanning Fluorimetry (DSF) 49

3.6.3 Circular Dichroism 51

3.6.4 Selwyn's Test 51

References 52

4 Buffers and their Use in the Study of Enzyme Mechanism 55

4.1 Introduction 55

4.1.1 Ionization and pK a 55

4.1.2 pH 57

4.1.3 Henderson-Hasselbalch Equation 57

4.1.4 Buffers 58

4.2 Buffering Capacity 60

4.3 Ionic Strength 60

4.4 Change in pH with Temperature 62

4.5 Choice of Buffer 63

4.6 Characteristics of Ionizing Groups in Proteins 65

4.7 Effect of pH on Enzyme Activity 66

4.7.1 Assumptions Required for the Analysis of pH Dependence 66

4.7.2 General Rate Equation for pH Dependence 67

4.7.3 Fitting pH Dependence 70

4.7.3.1 Double Ionizing Systems 70

4.7.3.2 Singularly Ionizing Systems 72

4.8 Effect of Solvent and Ionic Strength 73

References 73

5 Steady-state Assays and their Design 75

5.1 Introduction 75

5.2 The Pre-steady State 75

5.3 Steady-state Assays 77

5.4 Assay Development 78

5.4.1 Requirements for Method Development 79

5.4.2 Different Types of Enzymes Assays 79

5.4.2.1 Direct Continuous Assay 79

5.4.2.2 Indirect Assays 80

5.4.2.3 Discontinuous Indirect Assays 80

5.4.2.4 Continuous Indirect Assays 80

5.4.2.5 Coupled Assays 80

5.5 Blank Rates 81

5.6 The Assay Development Process 81

5.6.1 Initial Assay Scoping 81

5.6.2 Substrate Dependence 83

5.6.2.1 Non-Michaelian Kinetics 87

5.6.2.2 Multiple Substrates 89

5.6.3 Plate Type 92

5.7 Assay Optimization 93

5.7.1 Factorial Experimental Design (FED) 93

5.7.2 Coupling Enzyme Considerations 96

5.8 Kinetic Characterization 96

5.8.1 Substrate Concentration 96

5.9 Assay Adaptability 97

5.9.1 Tool Compounds 97

5.9.2 DMSO Tolerance 97

5.9.3 Assay Stability 97

5.9.4 Triage Assays 98

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