Thymidylate synthetase. Catalysis of dehalogenation of 5-bromo- and 5-iodo-2'-deoxyuridylate

Abstract

Thymidylate synthetase (EC 2.1.1.45) [from Lactobacillus casei] catalyzes the facile dehalogenation of 5-bromo-2''-deoxyuridylate (BrdUMP) and 5-iodo-2''-deoxyuridylate (IdUMP) to give 2''-deoxyuridylate (dUMP), the natural substrate of the enzyme. The reaction does not require folate cofactors and stoichiometrically consumes 2 equivalents of thiol. A minor product is also formed during the debromination of BrdUMP which was identified as a 5-alkylthio derivative formed by displacement of Br- by thiolate. The reaction proceeds with a substantial .alpha.-secondary inverse tritium isotope effect (kT/kH = 1.212-1.258) with [2-14C,6-3H]-BrdUMP as the substrate. An inverse tritium isotope effect of 1.18 was observed in the nonenzymatic chemical counterpart of this reaction, the cysteine-promoted dehalogenation of [2-14C,6-3H]-5-bromo-2''-deoxyuridine. Previous evidence for the mechanism of action of this enzyme has rested largely on chemical model studies and on information obtained from its stoichiometric interaction with the inhibitor 5-fluoro-2''-deoxyuridylate. The magnitude of the secondary isotope effect during the enzymatic dehalogenation described here provides direct proof for nucleophilic catalysis and formation of 5,6-dihydroprimidine intermediates in a reaction catalyzed by thymidylate synthetase.