THE STOICHIOMETRY OF THE CYTOCHROME P-450-CATALYZED METABOLISM OF METHOXYFLURANE AND BENZPHETAMINE IN THE PRESENCE AND ABSENCE OF CYTOCHROME B(5)

The complete stoichiometry of the metabolism of the cytochrome b(5) (c yt b(5))-requiring substrate, methoxyflurane, by purified cytochrome P -450 2B4 was compared to that of another substrate, benzphetamine, whi ch does not require cyt b(5) for its metabolism. Cyt b(5) invariably i mproved the efficiency of product formation. That is, in the presence of cyt b(5) a greater percentage of the reducing equivalents from NADP H were utilized to generate substrate metabolites, primarily at the ex pense of the side product, superoxide. With methoxyflurane, cyt b(5) a ddition always resulted in an increased rate of product formation, whi le with benzphetamine the rate of product formation remained unchanged , increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b(5). Un der some experimental conditions cyt b(5) inhibits total NADPH consump tion. Whether stimulation, inhibition, or no change in product formati on is observed in the presence of cyt b(5) depends on the net effect o f the stimulatory and inhibitory effects of cyt b(5). When total NADPH consumption is inhibited by cyt b(5), the rapidly metabolized, highly coupled (congruent to 50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the effi ciency (2-20%) of the reaction. In contrast, in the presence of the sl owly metabolized, poorly coupled (congruent to 0.5-3%) substrate, meth oxyflurane, inhibition of total NADPH consumption by cyt b(5) was neve r sufficient to overcome the stimulation of product formation due to a n increase in efficiency of the reaction.