Reconstitution of recombinant cytochrome P450 2C10(2C9) and comparison with cytochrome P450 3A4 and other forms: effects of cytochrome P450-P450 and cytochrome P450-b5 interactions.
Reconstitution of recombinant cytochrome P450 2C10(2C9) and comparison with cytochrome P450 3A4 and other forms: effects of cytochrome P450-P450 and cytochrome P450-b5 interactions.
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DOI：
10.1006/abbi.1997.0125
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发表时间：
1997-06
影响因子：
3.9
通讯作者：
H. Yamazaki;E. Gillam;M. Dong;William W. Johnson;F. Guengerich;T. Shimada
中科院分区：
文献类型：
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作者：
H. Yamazaki;E. Gillam;M. Dong;William W. Johnson;F. Guengerich;T. Shimada
在含有重组人细胞色素P450的系统中重建甲苯磺丁脲甲基羟基化和S-华法林7-羟基化活性（P450或CYP 1A 2）2C 10（2C 9），并与丁呋洛尔1 '-羟基化、茶碱8-羟基化、丁呋洛尔1'-羟基化、氯唑沙宗6-羟基化、以及通过CYP 3A 4的睾酮6 β-羟基化。CYP 2C 10需要细胞色素b5（b5）来实现甲苯磺丁脲和S-华法林氧化的最佳速率，b5可以被apo-b5取代; apo-b5和b5对重构系统的影响已经在含有CYP 3A 4的系统中报道，用于睾酮和硝苯地平的氧化以及用于通过NADPH-P450还原酶快速还原CYP 3A 4（H. Yamazaki等人，1996，J.Biol.Chem.271，27438-27444）。然而，停流研究表明，apo-b5以及b5没有引起刺激的还原CYP 2C 10的NADPH-P450还原酶，而还原率依赖于在重建系统的底物。氯唑沙宗6-羟基化的CYP 2 E1刺激b5，但不是由apo-b5，在重建系统。apo-和holo-b5均未增加CYP 1A 1或2D 6的丁呋洛尔1 '-羟基化活性或CYP 1A 2的茶碱8-羟基化活性。有趣的是，我们发现CYP 3A 4对睾酮6 β-羟基化的作用受到CYP 1A 2（以及天然人蛋白的前36个残基被去除的修饰形式）和CYP 1A 1以及b5的刺激，而当其他P450蛋白（例如，CYP 2C 10、2D 6或2 E1）加入复溶系统中。与此相反，CYP 2C 10和CYP 2 E1的底物氧化不受其他P450蛋白的刺激。目前的结果表明，有不同的最佳条件重建的底物氧化的各种形式的人P450酶，和在一些P450催化的反应蛋白质-蛋白质之间的相互作用P450和b5和其他P450蛋白质是非常重要的一些氧化催化的CYP 2C 10，2 E1和3A 4。
Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5; apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.