Methylenetetrahydrofolate reductase from Escherichia coli: Elucidation of the kinetic mechanism by steady-state and rapid-reaction studies

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherich ia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH2-H(4) folate) to 5-methyltetrahydrofolate (CH3-H(4)folate) using NADH as the sour ce of reducing equivalents. The enzyme also catalyzes the transfer of reduc ing equivalents from NADH or CH3-H(4)folate to menadione, an artificial ele ctron acceptor. Here, we have determined the midpoint potential of the enzy me-bound flavin to be -237 mV. We have examined the individual reductive an d oxidative half-reactions constituting the enzyme's activities. In an anae robic stopped-flow spectrophotometer, we have measured the rate constants o f flavin reduction and oxidation occurring in each half-reaction and have c ompared these with the observed catalytic turnover numbers measured under s teady-state conditions. We have shown that, in all cases, the half-reaction s proceed at rates sufficiently fast to account for overall turnover, estab lishing that the enzyme is kinetically competent to catalyze these oxidored uctions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin b y CH2-H(4)folate is substantially rate limiting in the physiological NADH-C H2-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the r eduction of flavin by CH3-H(4)folate in the CH3-H(4)folate-menadione oxidor eductase reaction. We conclude that studies of individual half-reactions ca talyzed by E. coli MTHFR may be used to probe mechanistic questions relevan t to the overall oxidoreductase reactions.