IDENTIFICATION OF RESIDUE-99, RESIDUE-220, AND RESIDUE-221 OF HUMAN CYTOCHROME-P450-2C19 AS KEY DETERMINANTS OF OMEPRAZOLE HYDROXYLASE-ACTIVITY

Human P450 2C19 is selective for 4'-hydroxylation of S-mephenytoin and 5-hydroxylation of omeprazole, while the structurally homologous P450 2C9 has low activity toward these substrates. To identify the critica l amino acids that determine the specificity of human P450 2C19, we co nstructed chimeras of P450 2C9 replacing various proposed substrate bi nding sites (SRS) with those of P450 2C19 and then replaced individual residues of P450 2C9 by site directed mutagenesis. The 339 NH2-termin al amino acid residues (SRS-1--SRS-4) and amino acids 160-383 (SRS-2-- SRS-5) of P450 2C19 conferred omeprazole 5-hydroxylase activity to P45 0 2C9. In contrast, the COOH terminus of P450 2C19 (residues 340-490 i ncluding SRS-5 and SRS-6), residues 228-339 (SRS-3 and SRS-4) and resi dues 292-383 (part of SRS-4 and SRS-B) conferred only modest increases in activity. A single mutation Ile(99) --> His increased omeprazole 5 -hydroxylase to similar to 51% of that of P450 2C19. A chimera spannin g residues 160-227 of P450 2C19 also exhibited omeprazole 5-hydroxylas e activity which was dramatically enhanced by the mutation Ile(99) --> His, A combination of two mutations, Ile(99) --> His and Ser(200) --> Pro, converted P450 2C9 to an enzyme with a turnover number for omepr azole 5-hydroxylation, which resembled that of P450 2C19. Mutation of Pro(221) --> Thr enhanced this activity. Residue 99 is within SRS-1, b ut amino acids 220 and 221 are in the F-G loop and outside any known S RS. Mutation of these three amino acids did not confer significant S-m ephenytoin 5-hydroxylase activity to P450 2C9, although chimeras conta ining SRS-1--SRS-4 and SRS-2--SRS-5 of P450 2C19 exhibited activity to ward this substrate. Our results thus indicate that amino acids 99, 22 0, and 221 are key residues that determine the specificity of P450 2C1 9 for omeprazole.