DOI

10.17077/etd.zi8f-qot8

Document Type

Dissertation

Date of Degree

Fall 2018

Access Restrictions

Access restricted until 01/31/2021

Degree Name

PhD (Doctor of Philosophy)

Degree In

Chemistry

First Advisor

Kohen, Amnon

Second Advisor

Quinn, Daniel M.

First Committee Member

Cheatum, Christopher M.

Second Committee Member

Forbes, Tori Z.

Third Committee Member

Shaw, Scott K.

Fourth Committee Member

Washington, Todd

Abstract

The functions of all living organism depend on thousands of biochemical reactions and enzymes catalyze most of these reactions necessary for biological life. A slight dysfunction of enzymatic activity in a cell can lead to a severe disease, even cell death. It is very important to understand the functions, mechanisms, and dynamics of enzymes for better drug development by targeting a specific enzyme. Thymidylate synthase (TSase) catalyzes the reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) to form 2′-deoxythymidine-5′-monophosphate (dTMP) utilizing N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate) as a cofactor. TSase is essential for synthesis of DNA and cellular division and thus maintains the intercellular thymidylate pool and as a result, TSase is a common target for many chemotherapeutic drugs. Throughout the complex mechanism catalyzed by TSase, there are two different C-H bond activations, a rate limiting hydride transfer and a much faster proton abstraction. This thesis describes studies that aim to understand the detailed mechanistic and dynamic features of TSase, using kinetic studies including kinetic isotope effects (KIEs), isotopic labelling of substrate and enzymes, site-directed mutagenesis, stopped-flow, quench-flow, structural biology, and QM/MM calculations as tools. In the TSase catalyzed reaction, the identity of the general base that abstracts the proton from the C-5 position of dUMP remains elusive. Crystallographic studies suggest that the active site consists of several conserved bases including Y94, E58, R166 and N177 (E. coli numbers). These residues are connected to each other via H-bonds coordinated by water molecules. To understand the role of specific residue of this H-bond network, we investigate the impact of mutations on catalysis using initial velocity and temperature dependence of KIE studies. Our findings suggest that a network of amino acids including Y94, R166 and several other amino acids and water assist to abstract the proton. A later chapter describes the trapping of a QM/MM proposed bi-substrate intermediate that consists of both dUMP and CH2H4folate moieties that forms upon enzymatic cysteine-substrate (S-C) bond breaking following the proton abstraction step. Currently, all the chemotherapeutic drugs targeting TSase either mimic the substrate dUMP or the cofactor folate, but no drug has combined both moieties. We have isolated and characterized the proposed bi-substrate intermediate by performing a rapid chemical quench of the E.coli TSase catalyzed reaction containing [2-14C]dUMP and [6-3H]MTHF using both high concentration of acid or base as a quencher. Detailed kinetics studies of the formation of this bi-substrate intermediate using QM/MM calculations also supports the formation of this intermediate as the predominant pathway. Our findings could potentially be instrumental for the development of new mechanism based inhibitor mimicking the bi-substrate intermediate. In another research, we have studied the role of fast dynamics on proton abstraction and hydride transfer steps in the TSase catalyzed reaction. We studied the effect of altered mass of the enzyme on different steps (hydride transfer and proton abstraction) catalyzed by a single enzyme, TSase. Comparisons are made of the steady-state kinetic parameters, temperature dependence of intrinsic kinetic isotope effects, and crystal structures of the light TSase, ‘heavy TSase’ (labeled with 13C, 15N and 2H) and ‘partial heavy’ TSase (labeled with 13C and 15N only). The results show a correlation between the mass perturbation of the protein and kcat, kcat/Km, and the difference in activation energy between isotopologues (ΔEa, T-H) for the hydride transfer step, whereas the ΔEa, T-H for the proton abstraction step remains unaffected by the isotopic substitution of TSase. The findings suggest that fast vibrations of the natural protein play a critical role in the fine-tuning of the narrowly distributed DADs at the transition state of hydride transfer, and yet do not play a central role in the fine-tuning of the more broadly distributed DADs at the transition state of the proton abstraction step. This distinction supports the possibility of selective involvement of fast vibrational frequencies in bond activation depending on the nature of the specific chemical step.

Pages

xvi, 126 pages

Bibliography

Includes bibliographical references (pages 112-126).

Copyright

Copyright © 2018 Ananda Kumar Ghosh

Available for download on Sunday, January 31, 2021

Included in

Chemistry Commons

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