Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies.
The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents. The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor. Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV. We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities. In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions. We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction. We conclude that studies of individual half-reactions catalyzed by E. coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions.
Trimmer
E E
EE
Department of Biological Chemistry and Biophysics Research Division, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Ballou
D P
DP
Matthews
R G
RG
D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D007700:Kinetics; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D008956:Models, Chemical; D009243:NAD / Q000737:chemistry; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry*; D013053:Spectrophotometry / Q000379:methods; D013763:Tetrahydrofolates / Q000737:chemistry; D014812:Vitamin K / Q000737:chemistry
D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D009243:NAD; D014812:Vitamin K; D005182:Flavin-Adenine Dinucleotide; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D016660:NAD(P)H Dehydrogenase (Quinone)