EXCLUSION OF 2'-DEOXYCYTIDINE 5'-MONOPHOSPHATE BY ASPARAGINE-229 OF THYMIDYLATE SYNTHASE

In thymidylate synthase (TS, EC 2.1.1.45), the only side chain in dire ct hydrogen bonding with the pyrimidine ring of the substrate dUMP is asparagine 229 (N229). In binary and ternary complexes, the carboxamid e moiety of the side chain of N229 forms a cyclic hydrogen bond networ k bridging N-3 and O-4 of the uracil heterocycle. Most of the N229 mut ants of TS bind dUMP and catalyze dTMP formation as well as the wild-t ype enzyme; thus, N229 does not contribute to binding of dUMP. Wild-ty pe TS binds dCMP weakly and does not accept dCMP as a substrate. Mutat ions at N229 of TS modify the interaction of TS with dCMP. TS N229D an d 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 an d 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 diffe rence 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 ur acil by Asn or Gln may account for specificity observed in other prote in-pyrimidine interactions.