Studies on the mechanism of formyltetrahydrofolate synthetase. The Peptococcus aerogenes enzyme

Two conflicting mechanisms have been proposed for formyltetrahydrofolate synthetase (EC 6.3.4.3). Detailed studies with a clostridial enzyme support a sequential mechanism, while a stepwise mechanism with formation of a dissociable intermediate has been proposed for the Peptococcus aerogenes synthetase. However, the data supporting the P. aerogenes mechanism were obtained using synthetase of questionable purity and the results supporting the mechanism could be attributed to contaminating activities. Consequently, uncertainty still exists with regard to the enzyme mechanism. To resolve this uncertainty, the P. aerogenes formyltetrahydrofolate synthetase has been purified to homogeneity and used in experiments to reinvestigate the reaction mechanism. The results of P1:ATP, ADP:ATP, and formate:10-formyltetrahydrofolate exchange experiments as well as a steady state kinetic analysis revealed no difference in the mechanisms of the P. aerogenes or clostridial synthetases. The results are inconsistent with a stepwise mechanism involving a dissociable intermediate and consistent only with a sequential mechanism.