RT Journal Article
SR Electronic
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
FD American Society for Pharmacology and Experimental Therapeutics
SP 1362
OP 1365
VO 29
IS 11
A1 Yu, Aiming
A1 Dong, Haijun
A1 Lang, Dieter
A1 Haining, Robert L.
YR 2001
UL http://dmd.aspetjournals.org/content/29/11/1362.abstract
AB 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