TY - JOUR T1 - Characterization of Dextromethorphan <em>O</em>- and<em>N</em>-Demethylation Catalyzed by Highly Purified Recombinant Human CYP2D6 JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 1362 LP - 1365 VL - 29 IS - 11 AU - Aiming Yu AU - Haijun Dong AU - Dieter Lang AU - Robert L. Haining Y1 - 2001/11/01 UR - http://dmd.aspetjournals.org/content/29/11/1362.abstract N2 - 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 fromCYP2D6*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 Km value of 1.9 ± 0.2 μM and a Vmax value of 8.5 ± 0.2 nmol/nmol of P450/min and measured simultaneously the formation of 3-methoxymorphinan with an apparentKm value of 5000 ± 700 μM andVmax 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. The American Society for Pharmacology and Experimental Therapeutics ER -