Document Type

Dissertation

Date of Degree

Spring 2015

Access Restrictions

Access restricted until 07/13/2018

Degree Name

PhD (Doctor of Philosophy)

Degree In

Chemistry

First Advisor

Amnon Kohen

Abstract

Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate bindings, chemical conversions, and products release. The relationship between protein motions and catalytic activity is of considerable contemporary interest in enzymology. To understand the factors influencing the rates of enzyme catalyzed reactions, the dynamics of the protein-solvent-substrate complex must be considered. The enzyme dihydrofolate reductase from Escherichia coli (EcDHFR) is often used as a model system in various biophysical studies, including those aimed at the examination of motions across the protein that may affect the catalyzed chemical transformation. Previously, molecular dynamics calculations, bioinformatics studies and intrinsic kinetic isotope effects (KIEs) of DHFR have suggested a network of coupled motions across the whole protein that is correlated to the reaction coordinate. This thesis describes studies that extend upon those initial results by studying the nature and extent of enzyme dynamics and motions in DHFR, both by using traditional experimental methods and by developing new biophysical probes of protein dynamics in this system. Kinetic techniques, site directed mutagenesis, methods involving isotopic labeling of substrates and proteins, immobilization techniques and molecular recognition force spectroscopy were combined to study EcDHFR. The major experimental methodology described in the subsequent chapters is the determination and analysis of intrinsic KIEs in a variety of EcDHFR mutants. The studies demonstrated that residues G121, M42 and F125, all of which are remote from the active site, FR participate in a network of coupled motions across the enzyme. Until recently, the missing link in our understanding of DHFR catalysis was the lack of a path by which such remote residues can affect the catalyzed chemistry at the active site. A later chapter describes studies that indicate synergism between a residue, G121, in that remote dynamic network and an active site residue, I14. In another related study all carbons and nitrogens, as well as non-exchangeable protons in EcDHFR were changed to their heavy isotopes (13C, 15N, 2H). This heavy enzyme has also been shown to be an efficient tool in addressing the heated debate regarding the role of protein dynamics in catalysis. Such enzyme generates a vibrationally perturbed "heavy ecDHFR", and the effect on the altered vibrations on catalysis was studied. Finally, the last two chapters describe techniques developed to immobilize DHFR on an AFM-mica chip via DNA linkers, concentration and activity assays of the immobilized enzyme, and single-molecule studies of the interaction between a tight inhibitor (methotrexate) and the enzyme. These studies reveal the distribution of states and interactions with ligands - a property not accessible for studies of a large ensemble of molecules. The high spatial and force resolution provided by AFM under physiological conditions have been utilized in this study to quantify the force-distance relationships of DHFR-methotrexate interactions. In the future, such an understanding of the interplay between enzyme function, structure and dynamics could eventually permit improved de novo construction of artificial biocatalysts, enable better inhibitor and drug design, and in general, further advance our ability to manipulate enzyme catalysis to our ultimate benefit.

Pages

xvi, 144 pages

Bibliography

Includes bibliographical references (pages 131-144).

Copyright

Copyright © 2015 Priyanka Singh

Available for download on Friday, July 13, 2018

Included in

Chemistry Commons

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