Role of Y94 in proton and hydride transfers catalyzed by thymidylate synthase

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 Escherichia coli, Y146 in Lactobacillus casei, and Y135 in humans) 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 turnover rate reduced by 2 orders of magnitude). 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 those for wt but, otherwise, were as temperature-independent, suggesting a perfectly preorganized reaction coordinate for both enzymes. At reduced temperatures, however, the KIE for the mutant increased with a decrease in temperature, indicating a poorly preorganized 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.