Formyltetrahydrofolate Synthetase: BINDING OF FOLATE SUBSTRATES AND KINETICS OF THE REVERSE REACTION

Abstract

Abstract During the fermentation of purines by Clostridium acidi-urici and Clostridium cylindrosporum, formyltetrahydrofolate synthetase is thought to catalyze the synthesis of ATP from (l)-10-formyltetrahydropteroyltriglutamate, ADP, and Pi. The reaction is energetically less favorable in this direction under conditions tested with the isolated enzyme. To further investigate the plausibility of this pathway, the kinetic parameters of the reaction in the direction of ATP synthesis were measured and the binding properties of the folate substrate and product, (l)-10-formyltetrahydropteroyltriglutamate and (l)-tetrahydropteroyltriglutamate, were investigated. Both folate coenzymes bind with a strong affinity to four identical, noninteracting sites per mole of enzyme. These sites are presumed to be the active sites, and their number is consistent with the tetrameric structure of the enzyme. The 10-formyl derivative also binds to additional sites, but with a lower affinity. This additional binding does not occur at the nucleotide binding site. Competition experiments were performed to determine the relative affinities of tetrahydrofolate and 10-formyltetrahydrofolate. The presence of the additional 2 γ-glutamyl residues results in approximately a 100-fold increase in binding affinity of the formylated and a 200-fold increase in binding affinity for the unformylated triglutamyl compounds. The unreduced pteroyltriglutamate was bound by the enzyme but with 10-fold lower affinity than the reduced derivative. Comparison of the Michaelis constants for 10-formyltetrahydrofolate and 10-formyltetrahydropteroyltriglutamate confirms the 100-fold difference in affinities. Both the mono- and triglutamyl derivatives were deformylated with the same maximal velocity, suggesting that the 2 additional γ-glutamyl residues are bound at a noncatalytic subsite. The results of kinetic experiments indicate that the enzyme can efficiently catalyze the synthesis of ATP and that the relative concentrations of formylated and unformylated folate coenzymes may be a significant regulatory parameter in the purine-fermenting Clostridia.