Abstract

Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262-to-arginine mutant of cytochrome P450 2B6 (P450 2B6.4) has been shown to have differential effects on P450 2B6 catalytic activity. We reported previously that the mutant enzyme was unable to metabolize 17-α-ethynylestradiol (17EE) or become inactivated by 17EE or efavirenz, which are inactivators of the wild-type enzyme. Studies were performed to elucidate the mechanism by which this mutation affects P450 2B6 catalytic activity. Studies using phenyldiazene to investigate differences between the active site topologies of the wild-type and mutant enzymes revealed only minor differences. Likewise, K_s values for the binding of both benzphetamine and efavirenz were comparable between the two enzymes. Using the alternate oxidant tert-butyl hydroperoxide, the mutant enzyme was inactivated by both 17EE and efavirenz. The stoichiometry of 17EE and efavirenz metabolism by P450s 2B6 and 2B6.4 revealed that the mutant enzyme was more uncoupled, producing hydrogen peroxide as the primary product. Interestingly, the addition of cytochrome b₅ improved the coupling of the mutant, resulting in increased catalytic activity. In the presence of cytochrome b₅ the variant readily metabolized 17EE and was inactivated by both 17EE and efavirenz. It is therefore proposed that the oxyferrous or iron-peroxo intermediate formed by the mutant enzyme in the presence of 17EE and efavirenz may be less stable than the same intermediates formed by the wild-type enzyme.