5,10-Methenyltetrahydrofolate synthetase. Purification and properties of the enzyme from rabbit liver.

The enzyme 5,10-methenyltetrahydrofolate synthetase has been purified to homogeneity from rabbit liver. The substrates are MgATP and 5-formyltetrahydrofolate. The enzyme will accept as substrates a variety of divalent metal ions and trinucleotides. The products of the reaction are 5,10-methenyltetrahydrofolate, ADP, and inorganic phosphate. The enzyme is a monomeric protein with a molecular weight of about 28,000. The stability of the enzyme is unusually sensitive to pH and ionic strength. Initial velocity studies suggest that the enzyme catalyzes a sequential mechanism. The turnover number of the enzyme is 300/min at 30 degrees C. The Km values for MgATP and 5-formyltetrahydrofolate are 0.3 mM and 0.5 microM, respectively. Product and dead-end inhibition studies indicate that substrates bind to the enzyme by a random mechanism. Vmax and the Km for MgATP are essentially constant over the pH range of 5.0-7.5. The results suggest that the enzyme catalyzes a reaction which serves as a salvage pathway to generate metabolically active one-carbon units from 5-formyltetrahydrofolate.