THYMIDYLATE SYNTHETASE CATALYZED EXCHANGE OF TRITIUM FROM [2'-DEOXYURIDYLATE-5-H-3 FOR PROTONS OF WATER

Thymidylate synthetase catalyzes an exchange of tritium of [5-3H]dUMP for protons of water in the absence of CH2-H4folate. The turnover number for this reaction is some 45 000-fold lower than that of dTMP formation and Km is 1.2 × 10-5 M, similar to the dissociation constant of the enzyme-dUMP complex determined by equilibrium dialysis. The presence of 1 mM folate has no effect on Vmax but results in a decrease in the Km of dUMP to a value close to that in the normal enzymic reaction. The exchange reaction provides definitive evidence that the enzymic reaction involves attack of a nucleophile of the enzyme on the 6 position of dUMP to provide a 5, 6-dihydro-dUMP intermediate which is covalently bound to the enzyme. Stereochemical considerations of the exchange reaction require proposal of a partial reaction which is not completely stereospecific or a complex reaction in which protons of water are handled with complete stereospecificity in a fashion similar to the one carbon unit of the normal enzymic reaction. © 1979, American Chemical Society. All rights reserved.