Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate (original) (raw)

Abstract

A study of the properties of the complex containing 5-fluoro-2'-deoxyuridylate (FdUMP), 5,lO-methylenetetrahydrofolate, and thymidylate synthetase is described. In the presence of the cofactor, isolable complexes contain two tightly bound molecules of FdUMP per enzyme molecule of 70,000 daltons. A number of folate analogs also stimulate binding of FdUMP, albeit to a lesser degree than the cofactor, Kinetic data indicate the rate constant for association of FdU.MP with the enzyme-methylenetetrahydrofolate complex to be 2 X lo7 M -~ min-l at 24". The unimolecular dissociation rates of FdUMP from the complex are highly temperature dependent and show AF* = 21.5 kcal/mol, AH* = 28.4 kcal/mol, and AS" = 0.023 eu; there is no indication that homotropic interactions, if existent, are manifested in the rate of dissociation of FdUMP. From kinetic data, an assocation constant for the interaction of FdUMP with the enzyme-cofactor complex is calculated to be ca. 2 x 10'0 M-' at 24 '. Within the enzyme-cofactor-FdUMP complex, a T hymidylate synthetase catalyzes the conversion of dUMP to TMP,' with concomitant transfer and reduction of the one carbon unit of CH2FAH4. In this process the hydrogen at C-6 of FAH4 is directly transferred to the methyl group of dUMP + 5,10-CHzFAH4 + TMP + FAHz TMP (Pastore and Friedkin, 1962) and 5-hydroxymethyl dUMP is not formed as an intermediate Flaks and Cohen, 1959).

Loading Preview

Sorry, preview is currently unavailable. You can download the paper by clicking the button above.