Role of Loop Motions in Enzyme Catalysis Pubblico

Reddish, Michael (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/4j03d0452?locale=it
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Abstract

Enzymes are important paradigms for studying catalysis. They are highly effective at increasing reaction rates with strong substrate specificity while operating at moderate conditions. Despite extensive characterization over the last century, the actual process of enzyme catalysis is not well understood. Simple investigations relating protein structure to function fail. These investigations tend to fail because they consider the enzyme as a static structure, ignoring the fact that proteins are known to exist as dynamic structures. Protein dynamics can occur on the order of small vibrations or large conformational rearrangements. One such dynamic feature common to many enzymes is the movement of loops throughout the catalytic cycle. Loop motion has typically been ill-studied due to experimental limitations in studying the microsecond to millisecond timescale of these motions. In this dissertation, we develop two complimentary approaches to studying this timescale, temperature-jump spectroscopy and microfluidic fast-mixing. We then apply these methods to study three different model enzyme systems: lactate dehydrogenase, dihydrofolate reductase, and purine nucleoside phosphorylase. By studying these enzymes we are able to establish that loop motions contribute a significant amount of heterogeneity to enzyme reactions requiring a reaction landscape, as opposed to the traditional reaction pathway, to fully describe the system. We then briefly discuss the implications of this approach for understanding enzymes in general.

Table of Contents

Chapter 1. Introduction to Enzyme Catalysis. 1

Chapter 2. Measuring Biomolecular Dynamics on the Submillisecond Timescale. 11

Section 2.1 Introduction to Common Submillisecond Techniques. 11

Section 2.2 Laser-Induced Temperature-Jump. 16

Section 2.3 Three-Dimensional Capillary Flow Fast Mixer. 23

Chapter 3. Lactate Dehydrogenase. 45

Section 3.1 Introduction to Lactate Dehydrogenase. 45

Section 3.2 Summary of Previous Catalytic Models. 48

Section 3.3 Infrared Temperature-Jump Experiments with LDH. 52

Section 3.4 Computational Modeling of Lactate Dehydrogenase Kinetics. 65

Section 3.5 Rapid Mixing Experiments with Competent and Inhibited Samples. 77

Section 3.6 Summary and Conclusions of LDH Work. 91

Chapter 4. Dihydrofolate Reductase. 94

Section 4.1 Introduction to Dihydrofolate Reductase. 94

Section 4.2 Temperature-Jump Studies on DHFR. 99

Section 4.3 Microfluidic Fast Mixing Applied to DHFR. 111

Chapter 5. Purine Nucleoside Phosphorylase. 115

Section 5.1 Introduction to Purine Nucleoside Phosphorylase. 115

Section 5.2 Microfluidic Fast Mixing Studies of PNP. 120

Chapter 6. Conclusion. 128

References. 131

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