The mechanism of the reaction catalyzed by N 10-formyltetrahydrofolate synthetase involves the formation of formyl phosphate as an intermediate which then formylates tetrahydrofolate at the N-10 position. Previous studies demonstrated that the non-enzymic formylation of tetrahydrofolate by formyl phosphate occurs exclusively at the more nucleophilic 5-nitrogen in the reduced pyrazine ring. The experiments described in this report were designed to determine whether N 5-formyltetrahydrofolate might be the first product to be formed on the enzyme, followed by formyl transfer to the 10-nitrogen via the cyclic intermediate n 5,10-methenyltetrahydrofolate. If this were the case, oxygen from solvent H2O would be incorporated into the formyl group of the N 10-derivative. By conducting the reaction in a 1:1 mixture of [16O]H2O and [18O]H2O and using 13C NMR spectroscopy we show that no 18O is incorporated into the product and conclude that the reaction proceeds via a direct formylation of the N-10 position by formyl phosphate.
