Kinetic isotope effects on the oxidation of reduced nicotinamide adenine dinucleotide phosphate by the flavoprotein methylenetetrahydrofolate reductase.
Previous work from this laboratory has established that the NADPH-menadione oxidoreductase reaction catalyzed by methylenetetrahydrofolate reductase from pig liver proceeds by Ping Pong Bi Bi kinetics and that the reductive half-reaction is rate limiting in steady-state turnover. We have now shown that methylenetetrahydrofolate reductase stereo-specifically removes the pro-S hydrogen from the 4-position of NADPH. During the oxidation of [4(S)-3H]NADPH, we observed a kinetic isotope on V/KNADPH of 10.8 +/- 0.4. When comparing the rates of oxidation of [4(S)-2H]NADPH and [4(S)-1H]NADPH, we measure kinetic isotope effects on V of 4.78 +/- 0.15 and on V/KNADPH of 4.54 +/- 0.59. When oxidation of [4(R)-2H]NADPH and [4(R)-1H]NADPH is compared, the secondary kinetic isotope effect on V is 1.04 +/- 0.01. When the NADPH-menadione oxidoreductase reaction is catalyzed in tritiated water, no incorporation of solvent tritium into residual NADPH is observed. We conclude from these observations that the oxidation of NADPH is largely or entirely rate limiting in the reductive half-reaction and, hence, in NADPH-menadione oxidoreductase turnover at saturating menadione concentration. In the presence of saturating NADPH, the flavin reduction proceeds with a rate constant of 160 S-1, which is at least 29-fold slower than estimates of the lower limit for the diffusion-limited rate constant characterizing NADPH binding to the enzyme under physiological conditions. Albery & Knowles have defined criteria for perfection in enzyme catalysis [Albery, W. J., & Knowles, J.R. (1976) Biochemistry 15, 5631-5640].(ABSTRACT TRUNCATED AT 250 WORDS)
D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D014812:Vitamin K / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases
D014812:Vitamin K; D009249:NADP; D006899:Mixed Function Oxygenases; D000429:Alcohol Oxidoreductases; D064417:Vitamin K Epoxide Reductases; D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2)