Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants

Experimental evidence for a 5-exocyclic methylene-dUMP intermediate in the thymidylate synthase reaction was recently obtained by demonstrating that t ryptophan 82 mutants of the Lactobacillus casei enzyme produced 5-(2-hydrox yethyl)thiomethyl-dUMP (HETM-dUMP) (Barret, J. E., Maltby, D. A., Santi, D. V., and Schultz, P. G. (1998) J. Am. Chem. Soc. 120, 449-450). The unusual product was proposed to emanate from trapping of the intermediate with bet a-mercaptoethanol in competition with hydride transfer from H(4)folate to f orm dTMP. Using mutants of the C-terminal residue of thymidylate synthase, we found that the ratio of HETM-dUMP to dTMP varies as a function of CH(2)H (4)folate concentration. This observation seemed inconsistent with the conc lusion that both products arose from a common intermediate in which CH(2)H( 4)folate was already bound to the enzyme. The enigma was resolved by a kine tic model that allowed for differential partitioning of the intermediate fo rmed on each of the two subunits of the homodimeric enzyme in forming the t wo different products. With three C-terminal mutants of L. casei TS, HETM-d UMP formation was consistent with a model in which product formation occurs upon occupancy of the first completely bound subunit, the rate of which is unaffected by occupancy of the second subunit. With one analogous E. coli TS mutant, HETM-dUMP formation occurred upon occupancy of the first subunit , but was inhibited when both subunits were occupied. With all mutants, dTM P formation occurs from occupied forms of both subunits at different rates; here, binding of cofactor to the first subunit decreased affinity for the second, but the reaction occurred faster in the enzyme form with both subun its bound to dUMP and CH(2)H(4)folate. The model resolves the apparent enig ma of the cofactor-dependent product distribution and supports the conclusi on that the exocyclic methylene intermediate is common to both HETM-dUMP an d dTMP formation.