Investigation on chemical steps of thymidylate synthase catalyzed reaction

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 proton abstraction 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 in the active site. Abstract Approved: ____________________________________ Thesis Supervisor ____________________________________ Title and Department ____________________________________ DateApproved: ____________________________________ Thesis Supervisor ____________________________________ Title and Department ____________________________________ Date INVESTIGATION ON CHEMICAL STEPS OF THYMIDYLATE SYNTHASE CATALYZED REACTION