Enzymatic activation of 5-formyltetrahydrofolate via conversion to 5, 10-methenyltetrahydrofolate.
The ATP-dependent conversion of 5-formyltetrahydrofolate (folinate) to the 5,10-methenyl derivative, catalyzed by 5,10-methenyltetrahydrofolate synthetase (EC 6.3.3.2), is of considerable importance in cancer chemotherapy, since it provides the basis for the administration of folinate to counteract the deleterious effects of high-dose Methotrexate regimens. Methenyltetrahydrofote synthetase has been purified 10,000-fold from L. casei using sequential affinity chromatography on immobilized folinate and ATP. The monomeric enzyme is homogeneous upon SDS-polyacrylamide gel electrophoresis, has a molecular weight of 23,000 (confirmed by gel filtration), and contains a single cysteine residue. The turnover number is ca. 250 min-1, and the Km values at pH 6 for 5-formyltetrahydrofolate and Mg-ATP are 0.6 and 1.0 microM, respectively; the equilibrium constant is 0.7-1.0 mM. Methotrexate, 5-methyltetrahydrofolate, and folate are not inhibitory. The mechanism for the reaction is proposed to involve phosphorylation of the formyl group to create an enol phosphate; subsequent attack on the methenyl carbon by N-10 would generate a tetrahedral intermediate, with release of the phosphate providing the driving force for ring closure.
D000818:Animals; D019731:Carbon-Nitrogen Ligases; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells / Q000201:enzymology; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D002955:Leucovorin / Q000378:metabolism*; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013763:Tetrahydrofolates / Q000378:metabolism*
D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase; D002955:Leucovorin