Methylenetetrahydrofolate reductase. Steady state and rapid reaction studies on the NADPH-methylenetetrahydrofolate, NADPH-menadione, and methyltetrahydrofolate-menadione oxidoreductase activities of the enzyme.
Methylenetetrahydrofolate reductase is a flavoprotein which has recently been purified to homogeneity from pig liver (Daubner, S. C., and Matthews, R. G. (1982) J. Biol. Chem. 257, 140-145). The enzyme catalyzes the transfer of reducing equivalents from NADPH to menadione or methylenetetrahydrofolate, or from methyltetrahydrofolate to menadione. We have now examined the individual reductive and oxidative half-reactions comprising these activities, measuring the rate of flavin reduction or oxidation in an anaerobic stopped flow apparatus. In all cases, the individual half-reactions occur at rates which are sufficiently fast to account for catalytic turnover, indicating that the enzyme is kinetically competent to catalyze these oxidoreductions by Ping Pong Bi Bi mechanisms. NADPH-linked reduction of the flavin appears to be rate-limiting for the NADPH-menadione oxidoreductase reaction, while reoxidation of the flavin is partially rate-limiting in the NADPH-methylenetetrahydrofolate oxidoreductase reaction. Reduction of the flavin by methyltetrahydrofolate is rate-limiting in the methyltetrahydrofolate-menadione oxidoreductase reaction. No intermediates are seen in any of these half-reactions. In agreement with our postulate of ping-pong mechanisms for the catalytic reactions of the enzyme is our observation of exchange of radiolabel between [methyl-14C]methyltetrahydrofolate and [methylene-14C]methylenetetrahydrofolate in the absence of either menadione or pyridine nucleotides. The rate of exchange when both substrates are saturating is in good agreement with the rate of the methyltetrahydrofolate-menadione oxidoreductase reaction measured under Vmax conditions.
D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D014812:Vitamin K / Q000378:metabolism
D014812:Vitamin K; D009249:NADP; D000429:Alcohol Oxidoreductases; D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases