DOI

10.17077/etd.58dron2h

Document Type

Dissertation

Date of Degree

Fall 2016

Access Restrictions

Access restricted until 02/23/2019

Degree Name

PhD (Doctor of Philosophy)

Degree In

Chemistry

First Advisor

Cheatum, Christopher M.

Second Advisor

Kohen, Amnon

First Committee Member

Kohen, Amnon

Second Committee Member

Cheatum, Christopher M.

Third Committee Member

Quinn, Daniel M.

Fourth Committee Member

Margulis, Claudio J.

Fifth Committee Member

Fuentes, Ernesto J.

Abstract

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.

Public Abstract

Enzymes are very important in our daily life. They are crucial catalyst for biological functions. Researchers have done tremendous works to understand how enzyme functions to either improve or disable an enzyme’s function so that it can be used in industry or for medical purposes. Enzyme studies also lead to development on artificial enzymes for special applications. The building blocks of an enzyme are amino acids, which need to fold properly to form the complex 3D structure. Comparing to the size of the entire macromolecule, the active site where chemistry happens is usually very small.

This macromolecule is not static. It is moving all the time, and motions involving different part of the enzyme take place at a wide range of time scales. For instance, movements associated with large domain of the enzyme happen at slower time scales, while localized motions happen at mush faster time scales. The chemistry step at the active site take place in the confined space, and the localized motions are fairly fast. In this work, I focus on the femtosecond (fs) to picosecond (ps) timescale motions and study whether and how this time scales dynamics affect or assist the catalysis.

The enzyme system under study is formate dehydrogenase (FDH), which enables the examination of the fs-ps dynamics using two-dimensional infrared spectroscopy and chemistry step kinetics using kinetic isotope effects (KIEs). Our work, for the first time, identifies the probable correlation between the fast protein dynamics and enzyme kinetics and provides a good fundamental working system for further studies.

Keywords

Enzyme Kinetics, Formate Dehydrogenase, Hydrogen Tunneling, Kinetic Isotope Effects, Protien Dynamics, Two-dimensional Infrared Spectroscopy

Pages

xvi, 122 pages

Bibliography

Includes bibliographical references (pages 113-122).

Copyright

Copyright © 2016 Qi Guo

Included in

Chemistry Commons

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