INFLUENCE OF AMINO-ACID RESIDUE-374 OF CYTOCHROME-P-450 2D6 (CYP2D6) ON THE REGIO-SELECTIVE AND ENANTIO-SELECTIVE METABOLISM OF METOPROLOL

Cytochrome P-450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme responsible for the oxidation of more than 30 widely used thera peutic agents. The enzymes encoded by the published genomic [Kimura, U meno, Skoda, Meyer and Gonzalez (1989) Am. J. Hum. Genet. 45, 889-904] and cDNA [Gonzalez, Skoda, Kimura, Umeno, Zanger, Nebert, Gelboin, Ha rdwick and Meyer (1988) Nature 331, 442-446] sequences of CYP2D6, and presumed to represent wild-type sequences, differ at residue 374 and e ncode valine (CYP2D6-Val) and methionine (CYP2D6-Met) respectively. Th e influence of this amino acid difference on cytochrome P-450 expressi on, ligand binding, catalysis and stereoselective oxidation of metopro lol was investigated by the heterologous expression of the correspondi ng cDNAs in the yeast Saccharomyces cerevisiae. The level of expressio n of apo- and hole-protein was similar with each form of CYP2D6 cDNA, and the binding affinities of a series of ligands to CYP2D6-Val and CY P2D6-Met were identical. The enantioselective O-demethylation and alph a-hydroxylation of metoprolol were also similar with each form of CYP2 D6, O-demethylation being R-(+)- enantioselective (CYP2D6-Val: R/S, 1. 6; CYP2D6-Met: R/S, 1.4), whereas alpha-hydroxylation showed a prefere nce for S-(-)-metoprolol (CYP2D6-Val: R/S, 0.7, CYP2D6-Met: R/S, 0.8). However, although the favoured regiomer overall was O-demethylmetopro lol (ODM), the regioselectivity for O-demethylation of each metoprolol enantiomer was significantly greater for CYP2D6-Val [R-(+)-: ODM/alph a-hydroxymetoprolol (alpha OH), 5.9; S-(-)-: ODM/alpha OH, 2.5) than t hat observed for CYP2D6-Met [R-(+)-: ODM/alpha OH, 2.2; S-(-)-: ODM/al pha OH, 1.4]. The stereoselective properties of CYP2D6-Val were consis tent with those observed for CYP2D6 in human liver microsomes. The dif ference in the stereoselective properties of CYP2D6-Val and CYP2D6-Met were rationalized with respect to a homology model of the active site of CYP2D6 based on an alignment with the crystal structure of the bac terial cytochrome P-450(BM-3), CYP102.