Homology models of cytochrome P450 2D6 (CYP2D6) have identified serine 304 as an active-site residue and implicated a putative role for this residue in substrate enantioselectivity and the differential inhibition of enzyme activity by the diastereoisomers quinine and quinidine. The role of serine 304 in selectivity is thought to be achieved through a preferential hydrogen-bond interaction between the hydroxyl group of the residue and one of the stereoisomers of each ligand. We have tested this hypothesis by substituting serine 304 with alanine, a non-hydrogen-bonding residue, and compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived enzyme. The Ser304Ala substitution did not alter the enantioselective oxidation of metoprolol; the O-demethylation reaction remained R-(+)-enantioselective (wild-type, RS, 1.7; mutant, RS, 1.6), whereas α-hydroxylation remained S-(-)-enantioselective (wild-type and mutant, R/S, 0.7). Similarly, the selective oxidation of the R-(+) and S-(-) enantiomers of propranolol to the major 4-hydroxy metabolite was identical with both wild-type and mutant forms of the enzyme (R/S 0.9), although the formation of minor metabolites (5-hydroxy and deisopropylpropranolol) did show some slight alteration in enantioselectivity. The differential inhibition of enzyme activity by quinine and quinidine was also identical with both forms of CYP2D6, the IC50 values for each enzyme being approx. 10 μM and 0.1 μM for quinine and quinidine, respectively. The kinetics of formation of α-hydroxymetoprolol and 4-hydroxydebrisoquine by wild-type and the Ser304Ala mutant was also very similar. However, modest changes in the regioselective oxidation of metoprolol and debrisoquine were observed with the Ser304Ala mutant. The regio- and enantioselective oxidation of an analogue of metoprolol, in which the hydroxyl group attached to the chiral carbon was replaced by a methyl moiety, was again identical with both wild-type and Ser304Ala mutant. However, the observed selectivity was the reverse of that observed with metoprolol. Collectively, these data indicate that Ser304 is unlikely to be a key ligand-binding residue, although the residue may indeed be located in the active-site cavity. The reversal of selectivity with the methyl analogue of metoprolol indicates that the hydroxyl group attached to the chiral centre of ligands, such as metoprolol, is important in defining the enzyme's selective properties, and that a hydrogen-bonding residue, other than Ser304, may be involved in this interaction. Current homology models of the active site of CYP2D6 that predict a hydrogen-bond interaction between Ser304 and specific ligands will need to be re-evaluated, and other candidate residues capable of such an interaction nominated and tested by site-directed mutagenesis studies.

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