THYMIDYLATE SYNTHETASE - CATALYSIS OF DEHALOGENATION OF 5-BROMO-2'-DEOXYURIDYLATE AND 5-IODO-2'-DEOXYURIDYLATE

Thymidylate synthetase 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 equiv of thiol. In addition to dUMP, a minor product is formed during the debromination of BrdUMP which has been identified as a 5-alkylthio derivative formed by displacement of bromide ion by thiolate. The reaction has been found to proceed with a substantial α-secondary inverse tritium isotope effect (kT/kH = 1.212-1.258) with [2-14C,6-3H]-BrdUMP as the substrate. Similarly, 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. © 1979, American Chemical Society. All rights reserved.