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) (cyt b(5))-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b(5) for its metabolism. Cyt b(5) invariably improved the efficiency of product formation. That is, in the presence of cyt b(5) a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b(5) addition always resulted in an increased rate of product formation, while 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). Under some experimental conditions cyt b(5) inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b(5) depends on the net effect of 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 efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (congruent to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b(5) was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.