18Oxygen incorporation into inorganic phosphate in the reaction catalyzed by N5,10-methenyltetrahydrofolate synthetase.

The mechanism of the N5,10-methenyltetrahydrofolate synthetase reaction was probed by determining the source of the oxygen atom introduced between the beta- and gamma-phosphates as ATP is converted to ADP and Pi. The reaction was performed using a mixture of [18O]- and [16O]N5-formyltetrahydrofolate in the presence of [16O]H2O and using [16O]N5-formyltetrahydrofolate in the presence of a 1:1 mixture of [18O]H2O and [16O]H2O. 31P NMR spectroscopy was used to examine the products. It was found that 18O from the formyl group was incorporated into Pi, and that 18O was not incorporated from the solvent. The results are consistent with a mechanism involving phosphorylation of the formyl group at the N5-position, followed by displacement of the phosphate by the 10-nitrogen.