Evidence that serine 304 is not a key ligand-binding residue in the activesite of cytochrome P450 2D6

Homology models of cytochrome P450 2D6 (CYP2D6) have identified serine 304 as an active-site residue and implicated a putative role for this residue i n substrate enantioselectivity and the differential inhibition of enzyme ac tivity by the diastereoisomers quinine and quinidine. The role of serine 30 4 in selectivity is thought to be achieved through a preferential hydrogen- bond interaction between the hydroxyl group of the residue and one of the s tereoisomers 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 Ser(304)Ala substitution did not alter the enantioselective oxidation of metoprolol; t he O-demethylation reaction remained R-(+)-enantioselective (wildtype, RIS, 1.7; mutant, RIS, 1.6), whereas alpha-hydroxylation remained S-( -)-enanti oselective (wild-type and mutant, RIS, 0.7). Similarly, the selective oxida tion of the R-(+) and S-(-) enantiomers of propranolol to the major 4-hydro xy metabolite was identical with both wild-type and mutant forms of the enz yme (RIS 0.9), although the formation of minor metabolites (5-hydroxy and d eisopropylpropranolol) did show some slight alteration in enantioselectivit y. The differential inhibition of enzyme activity by quinine and quinidine was also identical with both forms of CYP2D6, the IC50 values for each enzy me being approx. 10 mu M and 0.1 mu M for quinine and quinidine, respective ly. The kinetics of formation of alpha-hydroxymetoprolol and 4-hydroxydebri soquine by wild-type and the Ser(304)Ala mutant was also very similar. Howe ver, modest changes in the regioselective oxidation of metoprolol and debri soquine were observed with the Ser(304)Ala mutant. The regio- and enantiose lective oxidation of an analogue of metoprolol, in which the hydroxyl group attached to the chiral carbon was replaced by a methyl moiety, was again i dentical with both wild-type and Ser(304)Ala mutant. However, the observed selectivity was the reverse of that observed with metoprolol. Collectively, these data indicate that Ser(304) is unlikely to be a key ligand-binding r esidue, although the residue may indeed be located in the active-site cavit y. The reversal of selectivity with the methyl analogue of metoprolol indic ates 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 Ser(304), may be involved in this interaction. Current homology models of the active site of CYP2D6 t hat predict a hydrogen-bond interaction between Ser(304) and specific ligan ds will need to be re-evaluated, and other candidate residues capable of su ch an interaction nominated and tested by site-directed mutagenesis studies .