Role of Y94 in Proton and Hydride Transfers Catalyzed by Thymidylate Synthase

Abstract

Thymidylate synthase (TS) catalyzes the substitution of a carbon-bound proton in a uracil base by a methyl group to yield thymine in the de novo biosynthesis of this DNA base. The enzymatic mechanism involves making and breaking several covalent bonds. Traditionally, a conserved tyrosine (Y94 in E. coli, Y146 in L. casei, and Y135 in human) was assumed to serve as the general base catalyzing the proton abstraction. That assumption was examined here by comparing the nature of the proton abstraction using wild type (wt) E. coli TS (ecTS) and its Y94F mutant (with a two orders of magnitude reduced turnover rate). A subsequent hydride transfer was also studied using the wt and Y94F. The physical nature of both H-transfer steps was examined by determining intrinsic kinetic isotope effects (KIEs). Surprisingly, the findings did not suggest a direct role for Y94 in the proton abstraction step. The effect of this mutation on the subsequent hydride transfer was examined by a comparison of the temperature dependency of the intrinsic KIE on both the wt and the mutant. The intrinsic KIEs for Y94F at physiological temperatures were slightly smaller than for wt, but otherwise, were as temperature independent, suggesting a perfectly pre-organized reaction coordinate for both enzymes. At reduced temperature, however, the KIE for the mutant increased with decreasing temperature, indicating a poorly pre-organized reaction coordinate. Other kinetic and structural properties were also compared and the findings suggested that Y94 is part of a H-bond network that plays a critical role at a step between the proton and the hydride transfers, presumably the dissociation of H 4 folate from the covalently bound intermediate. The possibility that no single residue serves as the general base in question, but rather, that the whole network of H-bonds at the active site catalyzes proton abstraction, is discussed.Thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridylate (dUMP) with 5,10-methylene−5, 6, 7, 8-tetrahydrofolate (CH 2 H 4 folate), forming thymidine monophosphate (dTMP) and 7, 8-dihydrofolate (H 2 folate) (1). TS activity is essential to living organisms since it catalyzes the de novo synthesis of one of the DNA building blocks. Consequently, TS is a common target in cancer chemotherapy, antibiotic drugs, and gene therapy (2,3). The TS-catalyzed reaction has been elucidated in detail by a wide variety of kinetic, genetic, and structural methodologies (1,(4)(5)(6), which have shown that TS is a homodimer utilizing a half-of-the-sites-activity mechanism (7,8). Steady state measurements indicated a bi-bi ordered mechanism with substrate (dUMP) binding before the CH 2 H 4 folate (4,9). Kinetic and structural studies identified coherent protein motion that appear coupled to a hydride transfer step (10,11), which is rate limiting for the wt TS. † This work was supported by NIH R01 GM65368-01 and NSF CHE-0133117 to A.K. *Address correspondence to this author: Tel: 319-335-0234. Fax: 319-335-1270, Email: amn...

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