Klose T S, Ibeanu G C, Ghanayem B I, Pedersen L G, Li L, Hall S D, Goldstein J A
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Arch Biochem Biophys. 1998 Sep 15;357(2):240-8. doi: 10.1006/abbi.1998.0826.
Specificity of human CYP2C9 for two substrates, diclofenac and ibuprofen, was studied using chimeras and site-directed mutants of CYP2C9 and the highly related CYP2C19 expressed in Escherichia coli. Data were correlated with the presence of putative substrate recognition sites (SRS). A CYP2C19 chimera containing residues 228-340 (SRS 3 and 4) of 2C9 conferred both diclofenac hydroxylation and 2- and 3-hydroxylation of ibuprofen. The regiospecificity of this construct for metabolism of ibuprofen differed from that of CYP2C9 by favoring 2-hydroxylation over 3-hydroxylation. A CYP2C9 construct containing residues 228-340 of CYP2C19 lacked both diclofenac and ibuprofen hydroxylase activities. When residues 228-282 (containing SRS 3) of CYP2C9 were replaced by those of CYP2C19, the chimera retained appreciable activity for diclofenac and ibuprofen, and tolbutamide activity was inhibited by a specific CYP2C9 inhibitor, sulfaphenazole. This suggested that SRS 3 is not important in conferring specificity. CYP2C9 and CYP2C19 differ in five residues within the region 283-340 (within SRS 4). Mutations to analyze SRS 4 were made on a CYP2C19 chimera containing residues 228-282 of CYP2C9. A single I289N mutation conferred a dramatic increase in diclofenac hydroxylation and a small increase in ibuprofen 2-hydroxylation. A second mutation (N286S and I289N) increased diclofenac hydroxylation and conferred a dramatic increase in ibuprofen 2-hydroxylation. A V288E mutation did not increase activity toward either substrate and decreased activity toward the two substrates in combination with the I289N or the N286S, I289N mutants. Therefore residues 286 and 289 of CYP2C9 are important in conferring specificity for diclofenac and ibuprofen.
利用在大肠杆菌中表达的CYP2C9及其高度相关的CYP2C19的嵌合体和定点突变体，研究了人CYP2C9对两种底物双氯芬酸和布洛芬的特异性。数据与假定的底物识别位点（SRS）的存在相关。包含2C9的228-340位残基（SRS 3和4）的CYP2C19嵌合体赋予了双氯芬酸羟基化以及布洛芬的2-和3-羟基化能力。该构建体对布洛芬代谢的区域特异性与CYP2C9不同，它更倾向于2-羟基化而非3-羟基化。包含CYP2C19的228-340位残基的CYP2C9构建体缺乏双氯芬酸和布洛芬羟化酶活性。当CYP2C9的228-282位残基（包含SRS 3）被CYP2C19的相应残基取代时，该嵌合体对双氯芬酸和布洛芬仍保留可观的活性，并且甲苯磺丁脲活性被特异性CYP2C9抑制剂磺胺苯吡唑抑制。这表明SRS 3在赋予特异性方面并不重要。CYP2C9和CYP2C19在283-340区域（SRS 4内）的五个残基上存在差异。在包含CYP2C9的228-282位残基的CYP2C19嵌合体上进行了分析SRS 4的突变。单个I²⁸⁹N突变使双氯芬酸羟基化显著增加，布洛芬2-羟基化略有增加。第二个突变（N²⁸⁶S和I²⁸⁹N）增加了双氯芬酸羟基化，并使布洛芬2-羟基化显著增加。V²⁸⁸E突变对两种底物的活性均未增加，并且与I²⁸⁹N或N²⁸⁶S、I²⁸⁹N突变体组合时，对两种底物的活性降低。因此，CYP2C9的286和289位残基在赋予对双氯芬酸和布洛芬的特异性方面很重要。
