IDENTIFICATION OF RESIDUE-286 AND RESIDUE-289 AS CRITICAL FOR CONFERRING SUBSTRATE-SPECIFICITY OF HUMAN CYP2C9 FOR DICLOFENAC AND IBUPROFEN

Specificity of human CYP2C9 for two substrates, diclofenac and ibuprof en, was studied using chimeras and site-directed mutants of CYP2C9 and the highly related CYP2C19 expressed in Escherichia coli. Data were c orrelated with the presence of putative substrate recognition sites (S RS). A CYP2C19 chimera containing residues 228-340 (SRS 3 and 4) of 2C 9 conferred both diclofenac hydroxylation and 2- and 3-hydroxylation o f ibuprofen. The regiospecificity of this construct for metabolism of ibuprofen differed from that of CYP2C9 by favoring 2-hydroxylation ove r 3-hydroxylation. A CYP2C9 construct containing residues 228-340 of C YP2C19 lacked both diclofenac and ibuprofen hydroxylase activities. Wh en residues 228-282 (containing SRS 3) of CYP2C9 were replaced by thos e of CYP2C19, the chimera retained appreciable activity for diclofenac and ibuprofen, and tolbutamide activity was inhibited by a specific C YP2C9 inhibitor, sulfaphenazole. This suggested that SRS 3 is not impo rtant in conferring specificity. CYP2C9 and CYP2C19 differ in five res idues within the region 283-340 (within SRS 4). Mutations to analyze S RS 4 were made on a CYP2C19 chimera containing residues 228-282 of CYP 2C9. A single I289N mutation conferred a dramatic increase in diclofen ac hydroxylation and a small increase in ibuprofen a-hydroxylation. A second mutation (N286S and I289N) increased diclofenac hydroxylation a nd conferred a dramatic increase in ibuprofen a-hydroxylation. A V288E mutation did not increase activity toward either substrate and decrea sed activity toward the two substrates in combination with the I289N o r the N286S, I289N mutants. Therefore residues 286 and 289 of CYP2C9 a re important in conferring specificity for diclofenac and ibuprofen. ( C) 1998 Academic Press.