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

The O-demethylation of dextromethorphan to dextrorphan in humans is catalyz ed primarily by cytochrome P450 2D6 (CYP2D6). However, contrary to conventi onal wisdom, preparations of recombinant cytochrome P450 (P450) expressed f rom CYP2D6*1 cDNA also appear to produce significant amounts of 3-methoxy-m orphinan, the N-demethylated metabolite of dextromethorphan, when assayed i n vitro. We hypothesized that both pathways were intrinsic to 2D6 and here further examine the kinetics of formation using a highly purified preparati on of CYP2D6 in a reconstituted lipid system. Purified CYP2D6 protein with a measured molecular weight of 55772.0 (55769.6 Da predicted) was reconstit uted into an active, lipid-vesicle environment with purified rat cytochrome P450 reductase before the addition of substrate and NADPH. Reaction kineti cs were followed, and apparent Michaelis-Menten constants were determined f or 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 muM and a V-max value of 8.5 +/- 0.2 nmol/nmol of P450/min and measur ed simultaneously the formation of 3-methoxymorphinan with an apparent K-m value of 5000 +/- 700 muM and V-max value of 176 +/- 12 nmol (nmol of P450) (-1) min(-1). These results indicate that at least two distinct binding ori entations exist for dextromethorphan within the active site of CYP2D6.