The General Base for the Thymidylate Synthase Catalyzed Proton Abstraction

Abstract

Thymidylate synthase (TSase, EC 2.1.1.45) catalyzes the reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) to form 2′-deoxythymidine-5′-monophosphate (dTMP), one of the precursor of DNA bases. Many chemotherapeutic drugs target TSase as it is essential for the de novo biosynthesis of DNA in nearly all organisms including human. Although there are two different C-H bond activation throughout the mechanism, in contrast to the well resolved mechanism for the hydride transfer step, the identity of the general base that abstract the proton from the C-5 position of dUMP remains elusive. A careful investigation of the mechanistic feature including proton abstraction step of TSase is essential for the selective design of mechanism-based inhibitor. As the conserved tyrosine 94 (E. coli) is in close proximity to the C-5 of dUMP, we compared the temperature dependence of intrinsic KIEs for wild type and Y94F mutant, where the hydroxyl nearest the abstracted proton is missing. The results show (Figure 1: Arrhenius plots of intrinsic primary KIEs on the proton transfer for wild type and Y94F) that the intrinsic KIEs on proton abstraction step for Y94F are more temperature dependent in comparison to that of wild type. In the framework of phenomenological models1 developed to analyze such data, this indicates a substantial modulation of the transition state for that C-H activation. Hence the current findings indicate that Y94 is part of the reaction coordinate for the proton abstraction from the C-5 position of dUMP. Better understanding of TSase reaction mechanism could be instrumental in the rational design of mechanism-based inhibitors. This work was supported by NIH R01 GM065368 and NSF CHE1149023 to AK