kinetics suggest a ping-pong mechanism for methylenetetrahydrofolate reductase, or a ternary complex mechanism in which NADPH binding precedes 5,10-methylenetetrahydrofolate
Methylenetetrahydrofolate reductase presents reacts with both NADPH and 5,10-methylenetetrahydrofolate at the si face. The reaction involves successive hydride transfers to and from the flavin cofactor. NADP+ dissociates before 5,10-methylenetetrahydrofolate binds the reduced enzyme
methylenetetrahydrofolate reductase stereospecifically removes the pro-S hydrogen from the 4-position of NADPH. The oxidation of NADPH is largely or entirely rate limiting in the reductive half-reaction and in NADPH-menadione oxidoreductase turnover at saturating menadione concentration