Interaction of 1-(5-phospho-.beta.-D-arabinofuranosyl)-5-substituted-uracils with thymidylate synthetase: mechanism-based inhibition by 1-(5-phospho-.beta.-D-arabinofurosyl)-5-fluorouracil

Abstract
A number of 1-(5-phospho-.beta.-D-arabinosyl)-5-substituted-uracil (ara-UMP) were examined as inhibitors of dTMP synthetase [from Escherichia coli]. As reversible inhibitors, all were substantially less potent than their 2''-deoxyribosyl counterparts. In the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate), ara-FUMP [ara-fluoro-UMP] caused a 1st-order, time-dependent inactivation of the enzyme. At 0.degree. C, kinetic studies indicated a reversible Kd of 3.6 .mu.M for the ara-FUMP-CH2-H4folate complex, and k = 0.22 min-1 for the subsequent inactivation. Spectral studies of the complex and its behavior toward protein denaturants demonstrate that its structure and stoichiometry are directly analogous to those which have previously been described for FdUMP [fluoro-dUMP]. The significance of this finding with regard to prodrugs of ara-FU [5-fluorouracil arabinoside] and the potential of ara-FU as a chemotherapeutic agent are discussed.

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