Identification of residues 286 and 289 as critical for conferring substrate specificity of human CYP2C9 for diclofenac and ibuprofen
Identification of residues 286 and 289 as critical for conferring substrate specificity of human CYP2C9 for diclofenac and ibuprofen
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DOI：
10.1006/abbi.1998.0826
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发表时间：
1998-09-15
影响因子：
3.9
通讯作者：
Goldstein, JA
中科院分区：
文献类型：
--
作者：
Klose, TS;Ibeanu, GC;Goldstein, JA
使用CYP 2C 9的嵌合体和定点突变体以及在大肠杆菌中表达的高度相关的CYP 2C 19，研究了人CYP 2C 9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。含有2C 9残基228-340（SRS 3和4）的CYP 2C 19嵌合体赋予双氯芬酸羟基化以及布洛芬的2-和3-羟基化。该结构对布洛芬代谢的区域特异性与CYP 2C 9不同，其有利于2-羟基化而不是3-羟基化。含有CYP 2C 19残基228-340的CYP 2C 9构建体缺乏双氯芬酸和布洛芬羟化酶活性。当CYP 2C 9的残基228-282（含SRS 3）被CYP 2C 19的残基取代时，嵌合体保留了对双氯芬酸和布洛芬的明显活性，甲苯磺丁脲活性被特异性CYP 2C 9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。CYP 2C 9和CYP 2C 19在区域283-340（SRS 4内）内的5个残基上存在差异。在含有CYP 2C 9残基228-282的CYP 2C 19嵌合体上进行突变以分析SRS 4。一个单一的I289 N突变赋予双氯芬酸羟基化的显着增加和布洛芬α-羟基化的小幅增加。第二个突变（N286 S和I289 N）增加了双氯芬酸羟基化，并使布洛芬α-羟基化显著增加。V288 E突变不增加对任一底物的活性，并且与I289 N或N286 S、I289 N突变体组合降低对两种底物的活性。因此，CYP 2C 9的残基286和289在赋予双氯芬酸和布洛芬特异性方面很重要。(C)北京：科学出版社.
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 a-hydroxylation. A second mutation (N286S and I289N) increased diclofenac hydroxylation and conferred a dramatic increase in ibuprofen a-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. (C) 1998 Academic Press.