Exclusion of 2'-deoxycytidine 5'-monophosphate by asparagine 229 of thymidylate synthase.

Abstract

In thymidylate synthase (TS, EC 2.1.1.45), the only side chain in direct hydrogen bonding with the pyrimidine ring of the substrate dUMP is asparagine 229 (N229). In binary and ternary complexes, the carboxamide moiety of the side chain of N229 forms a cyclic hydrogen bond network bridging N-3 and O-4 of the uracil heterocycle. Most of the N229 mutants of TS bind dUMP and catalyze dTMP formation as well as the wild-type enzyme; thus, N229 does not contribute to binding of dUMP. Wild-type TS binds dCMP weakly and does not accept dCMP as a substrate. Mutations at N229 of TS modify the interaction of TS with dCMP. TS N229D and TS N229E catalyze the methylation of dCMP [Liu, L., & Santi, D. V. (1992) Biochemistry 31, 5010-5014]. With the exception of the TS N229Q, most of the N229 mutants bind dCMP as well as or tighter than dUMP and bind dCMP 300-3000-fold tighter than wild-type TS. We conclude that TS discriminates binding of dUMP versus dCMP by a 3-4 kcal mol-1 difference in binding energy by exclusion of dCMP from the active site. We propose that this exclusion is a consequence of untoward interactions between dCMP and the side-chain carboxamide group of the Asn or Gln at position 229 of TS. We speculate that exclusion of cytosine versus uracil by Asn or Gln may account for specificity observed in other protein-pyrimidine interactions.