Characterization of dextromethorphan O- and N-demethylation catalyzed by highly purified recombinant human CYP2D6

The O-demethylation of dextromethorphan to dextrorphan in humans is catalyzed primarily by cytochrome P450 2D6 (CYP2D6). However, contrary to conventional wisdom, preparations of recombinant cytochrome P450 (P450) expressed from CYP2D6*1 cDNA also appear to produce significant amounts of 3-methoxymorphinan, the N-demethylated metabolite of dextromethorphan, when assayed in vitro, We hypothesized that both pathways were intrinsic to 2D6 and here further examine the kinetics of formation using a highly purified preparation of CYP2D6 in a reconstituted lipid system. Purified CYP2D6 protein with a measured molecular weight of 55772.0 (55769.6 Da predicted) was reconstituted into an active, lipid-vesicle environment with purified rat cytochrome P450 reductase before the addition of substrate and NADPH. Reaction kinetics were followed, and apparent Michaelis-Menten constants were determined for the appearance of each metabolite by high-pressure liquid chromatography, using both UV and fluorescence detection. In a 2-min assay, purified 2D6 catalyzed the formation of dextrorphan with an apparent K_m value of 1.9 ± 0.2 μM and a V_max value of 8.5 ± 0.2 nmol/nmol of P450/min and measured simultaneously the formation of 3-methoxymorphinan with an apparent Km value of 5000 ± 700 μM and V_max value of 176 ± 12 nmol (nmol of P450)^-1 min^-1. These results indicate that at least two distinct binding orientations exist for dextromethorphan within the active site of CYP2D6.