Identification of human CYP2C19 residues that confer S-mephenytoin 4 '-hydroxylation activity to CYP2C9

CYP2C19 is selective for the 4 ' -hydroxylation of S-mephenytoin while the highly similar CYP2C9 has We activity toward this substrate. To identify cr itical amino acids determining the specificity of human CYP2C19 for S-mephe nytoin 4 ' -hydroxylation, we constructed chimeras by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagen esis. Only a chimera containing regions encompassing SRSs 1-4 was active (3 0% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six r esidues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin 4 ' hydroxylase (6% of the activity of wild-typ e CYP2C19). Of these, only the I99H difference located in SRS 1 between hel ices B and C reflects a change in a side chain that is predicted to be in t he substrate-binding cavity formed above the heme prosthetic group. Two add itional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five di fferences in the N-terminal portion of helix I conferred S-mephenytoin 4 ' -hydroxylation activity with a KM similar to that of CYP2C19 but a 3-fold l ower K-cat. Three residues in helix I, S286N, V292A, and F2951, were essent ial for S-mephenytoin 4 ' -hydroxylation activity. On the basis of the stru cture of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influ ence the packing of substrate binding site residues and likely substrate ac cess channels in the enzyme.