DOI

10.17077/etd.1x7qyras

Document Type

Dissertation

Date of Degree

Spring 2017

Access Restrictions

Access restricted until 07/13/2019

Degree Name

PhD (Doctor of Philosophy)

Degree In

Chemistry

First Advisor

Cheatum, Christopher M.

Second Advisor

Kohen, Amnon

First Committee Member

Wiemer, David

Second Committee Member

Quinn, Daniel

Third Committee Member

Elcock, Adrian

Abstract

Enzyme dynamics occur on a wide range of length and timescales. This work is focused on understanding enzyme dynamic at the fs-ps timescale as this is the dynamic range at which bonds are typically made and broken during chemical reactions. Our work focuses on enzymes that catalyze hydride transfer between two carbon atoms - a fundamental reaction in biology. Primary kinetic isotope effects and their temperature dependence have implied that fast dynamics of the enzyme are important in facilitating hydride transfer, however these experiments do not measure any such motions directly. We make use of two-dimensional infrared spectroscopy (2D IR), a technique that interrogates the vibrations of molecules to extract dynamic information from the surrounding environment with 100 fs resolution. A model system, formate dehydrogenase (FDH), is an excellent probe of dynamics at the fs-ps timescale. Azide bound to the ternary complex of FDH offers the ability to measure dynamics of an analog structure of the reactive complex using 2D IR, while also studying the reaction directly with and KIE’s and their temperature dependence. By altering various parts of the structure of FDH via mutagenesis and other techniques, we investigate the role of structure and dynamics to determine how fast dynamics of the active site influence the the kinetics of hydride transfer. These experiments are the first means of providing a dynamic interpretation of KIEs and their temperature dependence.

Public Abstract

Enzymes catalyze chemical reactions that are fundamental in all biological systems. Without them nearly all biochemical reactions would not occur on a time scale relevant for life to exist. Enzymes move on a variety of length and time scales: ms-s fluctuations of the enzyme are involved in large conformational changes that may bind and release substrates, ns-ms motions are involved in torsional motions of amino acid -R groups and local structural fluctuations. Even faster still are bond rotations and vibrations that occur on the fs-ps time scale. It is these very fast motions that our research is primarily focused on, as this is the time scale at which collisions occur in solution and is therefore likely that this is the time scale at which bonds are made and broken during enzyme catalyzed chemical reactions. Understanding if enzyme motions at this time scale are relevant to promoting chemical reactions is the major focus of this research.

Keywords

2D Infrared Spectroscopy, Catalysis, Kinetic Isotope Effect, Tunneling

Pages

x, 79 pages

Bibliography

Includes bibliographical references (pages 73-79).

Copyright

Copyright © 2017 Philip Lee Pagano Jr.

Included in

Chemistry Commons

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