Document Type

Dissertation

Date of Degree

2007

Degree Name

PhD (Doctor of Philosophy)

Degree In

Chemistry

First Advisor

Amnon Kohen

Abstract

Thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridylate (dUMP) with cofactor 5,10-methylene-5, 6, 7, 8-tetrahydrofolate, forming thymidine monophosphate (dTMP) and 7, 8-dihydrofolate. The reaction pathway is comprised of at least seven covalent bond cleavage or formation, therefore, it is a very complex system for scientists to explore the mechanistic details of this reaction. In my Ph.D work, two microscopic steps in TS-catalyzed pathway were investigated by means of kinetic isotope effects (KIE), their temperature dependence and steady state initial velocity measurements. Chapter II of my thesis focuses on testing the quantum mechanical behavior of the last hydride transfer step. Chapter III is involved in studying the coupled motion between primary and secondary hydrogens for the same hydride transfer step. In Chapter IV we used same methodology to explore the mechanistic details on the last hydrogen transfer step by comparison of the temperature dependency of the intrinsic KIE using the wild type and the mutant W80M TS. The effect of the mutation on the subsequent hydride transfer was examined by comparison of the temperature dependency of the intrinsic KIE using the wild type and Y94F TS. The further examination of the subsequent hydride transfer using the wt and Y94F gave details on the reaction mechanism and on the role of tyrosine (a highly conserved residue) in the active site.

Pages

ix, 114 pages

Bibliography

Includes bibliographical references (pages 108-114).

Copyright

Copyright 2007 Baoyu Hong

Included in

Chemistry Commons

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