The Stoichiometry of the Cytochrome P-450-catalyzed Metabolism of Methoxyflurane and Benzphetamine in the Presence and Absence of Cytochrome b5

Abstract

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 (Х50%) 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 (Х0.5-3%) 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.

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

“…Wild type cyt b 5 caused a 6-fold stimulation of the rate of fluoride formation from 0.21 Ϯ 0.03 nmol of F Ϫ min Ϫ1 nmol of cyt P450 Ϫ1 in the absence of cyt b 5 to 1.23 Ϯ 0.2 nmol min Ϫ1 nmol of cyt P450 Ϫ1 with cyt b 5 . These results are similar to the rate of methoxyflurane metabolism observed in previous studies (7,9). Replacing the conserved residues Pro 95 and Lys 94 with alanines and reversing the amino acid sequence of the linker region did not significantly alter the rate of F Ϫ formation.…”

“…It is unlikely that mutation of the cyt b 5 linker region alters the relative formation of the two products, as recent modeling studies have shown that, in cyt P450 2E1, the preferential metabolism of methoxyflurane to methoxydifluroacetic acid is the result of differences in the relative activation energy required for H atom extraction rather than favorable substrate binding conformations (31). In addition, the relative amounts of methoxyflurane products formed by cyt P450 2B4 are the same in the presence and absence of cyt b 5 (7). These studies indicate that the observed reduction in F Ϫ formation caused by mutation of the cyt b 5 linker region is the result of an attenuation in the overall metabolism of methoxyflurane rather than a selective decrease in O-demethylation relative to dechlorination.…”

“…5 -The method used to measure the rate of methoxyflurane metabolism by cyt P450 2B4 in the presence and absence of cyt b 5 is a modification of a previously described procedure (7). The reaction components were added to a reaction mixture in a specific sequence: 50 mM KPO 4 , pH 7.4, 10 M cyt P450 2B4, 10 M cyt P450 reductase, 0 -90 M cyt b 5 , and 1.9 mM DLPC.…”

“…However, cyt b 5 can also inhibit the rate of oxidation catalyzed by cyt P450, by competing with cyt P450 reductase. This effect of cyt b 5 is observed as a decrease in the overall rate of NADPH consumption (7).…”

Reduced Oxy Intermediate Observed in D251N Cytochrome P450cam

“…Some of the products formed by these enzymes have also been implicated as causative agents in a number of cancers (Gonzales & Gelboin, 1994;Shimada et al, 1996). Intermediates involved in dioxygen activation have been proposed (Fruetel et al, 1992, Gruenke et al, 1995, Loida & Sligar, 1993 from the production of superoxide or peroxide upon loss of catalytic efficiency. The mechanism of dioxygen activation is the least understood step in P450 catalysis (Figure 1), which begins with the reduction of the dioxygen complex (oxy-P450, 4 in Figure 1).…”