Additional experiments were carried out to elucidate the mechanism of the reaction catalyzed by crystalline formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. The ratio of formate kinase and formyltetrahydrofolate synthetase activities, compared during the purification of the latter enzyme, was found to vary. It is possible to prepare formyltetrahydrofolate synthetase free of formate kinase activity. Kinetic studies show that the affinity constant of any substrate is not affected by variation of the concentration of the other two substrates. Incubation of stoichiometric amounts of the enzyme with formate-C14 and ATP failed to indicate any interaction between the enzyme and these two substrates. The reversibility of the reaction was demonstrated by measuring the disappearance of 10-formyltetrahydrofolate in the presence of orthophosphate and ADP. Unusually large amounts of enzyme were required to demonstrate the reaction in this direction compared to the amount of enzyme required to demonstrate the formation of 10-formyltetrahydrofolate. The exchange of formate-C14 into 10-formyltetrahydrofolate requires the presence of both ADP and orthophosphate. These results are consistent with the "concerted" mechanism proposed previously.
|Original language||English (US)|
|Number of pages||8|
|Journal||Archives of Biochemistry and Biophysics|
|Publication status||Published - Sep 1964|
ASJC Scopus subject areas
- Molecular Biology