Catalytic turnover of pyrene by CYP3A4: Evidence that cytochrome b5 directly induces positive cooperativity

Abstract

The metabolism of pyrene to hydroxypyrene by CYP3A4 was investigated to determine the effect of cytochrome b 5 ( b 5) on turnover kinetics. In the absence of b 5, formation of hydroxypyrene in in vitro incubations showed a biphasic substrate–velocity curve where K m1 and V max1 were 1.3 μM and 0.5 pmol/min/pmol P450, respectively. The addition of testosterone to the incubation mixture completely abolished the second phase to yield a typical, hyperbolic curve, presumably through the disruption in the formation of a π–π stacked pyrene complex within the CYP3A4 active site. Finally, the addition of b 5 yielded an increase hydroxypyrene formation that resulted in a sigmoidal substrate velocity curve. The V max was 15.7 pmol/min/pmol P450, the K m was 7.5 μM, and the Hill coefficient was greater than two. This demonstrated that b 5 could directly induce positive cooperativity on CYP3A4 and that this biological factor needs to be carefully considered when included in in vitro P450 reactions.