On the structure of the cofactor in the complex formed with thymidylate synthetase, 5,10-methylenetetrahy-drofolate and 5-fluoro-2'-deoxyuridylate.

A study was undertaken to ascertain whether dihydrofolic acid is produced in the complex formed with 5,10-methylenetetrahydrofolate, 5-fluorodeoxyuridylate and thymidylate synthetase, as suggested by ultraviolet difference spectral studies. The complex was formed using the cofactor specifically labeled with tritium at the 6-position. After dissociation by equilibration with unlabeled cofactor, it was demonstrated that the tritium remained exclusively at the 6-position. Had oxidation of the cofactor occurred within the complex to give a methylated enzyme form, tritium should have been transferred to the one-carbon unit of the cofactor. It was also found that the difference spectrum of the ternary complex which resembles that of dihydrofolic acid can also be produced by substituting an analog of the cofactor which is not susceptible to oxidation. The results described here demonstrate that oxidation of the cofactor does not occur in the ternary complex and suggest that the unusual ultraviolet spectrum results from perturbations of a chromophore of the bound cofactor.