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 microM 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 K(m) value of 5000 +/- 700 microM 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.
Yu
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Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506-9530.
Dong
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Lang
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Haining
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003915:Dextromethorphan / Q000737:chemistry / Q000493:pharmacokinetics*; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry / Q000493:pharmacokinetics*; D006801:Humans; D007700:Kinetics; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010090:Oxidoreductases, O-Demethylating / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D018691:Excitatory Amino Acid Antagonists; D011994:Recombinant Proteins; D003915:Dextromethorphan; D010090:Oxidoreductases, O-Demethylating; D019389:Cytochrome P-450 CYP2D6; D010089:Oxidoreductases, N-Demethylating