Influence of phenylalanine-481 substitutions on the catalytic activity of cytochrome P450 2D6

Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have ide ntified phenylalanine 481 (Phe(481)) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in pi-pi interactions with the benzene ring of ligands. We have tested this hypothe sis by replacing Phe(481) with tyrosine (Phe(481) --> Tyr), a conservative substitution, and with leucine (Phe(481), Leu) or glycine (Phe(481) --> Gly ), two non-aromatic residues, and have compared the properties of the wild- type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived protein. The Phe(481) --> Tyr substitution did not alter the kinetics [K-m (muM) and V-max (pmol/min per pmol) respective ly] of oxidation of S-metoprolol (27; 4.60), debrisoquine (46; 2.46) or dex tromethorphan (2; 8.43) relative to the respective wild-type values [S-meto prolol (26; 3.48), debrisoquine (51; 3.20) and dextromethorphan (2: 8.16)]. The binding capacities K-s (muM)] of a range of CYP2D6 ligands to the Phe( 481) --> Tyr enzyme (S-metoprolol, 22.8; debrisoquine, 12.5; dextromethorph an, 2.3; quinidine, 0.13) were also similar to those for the wild-type enzy me (S-metoprolol, 10.9; debrisoquine, 8.9; dextromethorphan, 3.1; quinidine , 0.10). In contrast, the Phe(481) --> Leu and Phe(481) --> Gly substitutio ns increased significantly (3-16-fold) the K-m, values of oxidation of the three substrates [S-metoprolol (12-124 muM), debrisoquine (152-184 muM) and dextromethorphan (20-31 muM)]. Similarly, the K-s values of the ligands to Phe(481) --> Leu and Phe(481) --> Gly mutants were also increased 3 to 10- fold (S-metoprolol, 33.2-41.9 muM; debrisoquine, 85-90 muM; dextromethorpha n, 15.7-18.8 muM; quinidine 0.35-0.53 muM). However, contrary to a recent p roposal that Phe(481) has the dominant role in the binding of substrates th at undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe(481 ) --> Gly substitution did not substantially decrease the capacity of the e nzyme to N-deisopropylate metoprolol (wild-type, 1.12 pmol/min per pmol of P450; Phe(481) --> Gly, 0.71), whereas an Asp(301) --> Gly substitution dec reased the N-dealkylation reaction by 95% of the wild-type rate. Overall. o ur results are consistent with the proposal that Phe(481) is a ligand-bindi ng residue in the active site of CYP2D6 and that the residue interacts with ligands via a pi-pi interaction between its phenyl ring and the aromatic m oiety of the ligand. However, the relative importance of Phe(481) in bindin g is ligand-dependent; furthermore, its importance is secondary to that of Asp(301). Finally, contrary to predictions of a recent homology model, Phe( 481) does not seem to have a primary role in CYP2D6-mediated N-dealkylation .