Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
Amino Acids. 2011 Apr;40(4):1195-204. doi: 10.1007/s00726-010-0743-8. Epub 2010 Sep 17.
The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of drugs and exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver. Nearly all previous works about polymorphic variants of CYP2C8 were focused on unpurified proteins, either cells or human liver microsomes; therefore their structure-function relationships were unclear. In this study, two polymorphic enzymes of CYP2C8 (CYP2C8.4 (I264M) and CYP2C8 P404A) were expressed in E. coli and purified. Metabolic activities of paclitaxel by the two purified polymorphic enzymes were observed. The activity of CYP2C8.4 was 25% and CYP2C8 P404A was 30% of that of WT CYP2C8, respectively. Their structure-function relationships were systematically investigated for the first time. Paclitaxel binding ability of CYP2C8.4 increased about two times while CYP2C8 P404A decreased about two times than that of WT CYP2C8. The two polymorphic mutant sites of I264 and P404, located far from active site and substrate binding sites, significantly affect heme and/or substrate binding. This study indicated that two important nonsubstrate recognition site (SRS) residues of CYP2C8 are closely related to heme binding and/or substrate binding. This discovery could be valuable for explaining clinically individual differences in the metabolism of drugs and provides instructed information for individualized medication.
细胞色素 P450（CYP）超家族在广泛的药物和外源性化学物质的氧化代谢中起着关键作用。CYP2C8 是人类肝脏中负责代谢抗癌药物紫杉醇的主要酶。几乎所有以前关于 CYP2C8 多态性变体的工作都集中在未纯化的蛋白质上，无论是细胞还是人肝微粒体；因此，它们的结构-功能关系尚不清楚。在这项研究中，两种 CYP2C8 的多态性酶（CYP2C8.4（I264M）和 CYP2C8 P404A）在大肠杆菌中表达并纯化。观察了两种纯化的多态性酶对紫杉醇的代谢活性。CYP2C8.4 的活性分别为 WT CYP2C8 的 25%和 CYP2C8 P404A 的 30%。他们的结构-功能关系首次得到系统研究。紫杉醇与 CYP2C8.4 的结合能力增加了约两倍，而 CYP2C8 P404A 则降低了约两倍，而与 WT CYP2C8 相比。I264 和 P404 的两个多态性突变位点，位于远离活性位点和底物结合位点的位置，显著影响血红素和/或底物结合。这项研究表明，CYP2C8 的两个重要非底物识别位点（SRS）残基与血红素结合和/或底物结合密切相关。这一发现可以为解释药物代谢中的个体差异提供有价值的信息，并为个体化用药提供指导信息。
