一种细胞色素P450 2C8基因多态—CYP2C8.3的结构和功能  

Structural and functional insights into CYP2C8.3:A genetic polymorph of cytochrome P450 2C8

作　　者：蒋华麟[1] 孙璐[1,2] 黄仲贤[1] 谭相石[1] 

机构地区：[1]金属蛋白和生物金属研究中心复旦大学化学系和生物医学研究院,上海200433 [2]第四军医大学第一附属医院心血管内科,西安710032

出　　处：《中国科学：化学》2011年第1期57-65,共9页SCIENTIA SINICA Chimica

基　　金：国家自然科学基金(20771029);上海市浦江人才计划(08PJ14017);上海市重点学科基金项目(B108)资助

摘　　要：细胞色素P450超级家族在代谢众多的外源性化学物质方面发挥重要的作用.细胞色素P4502C8是人体肝脏中主要负责代谢抗癌药物紫杉醇的酶,它至少负责代谢5%的临床药物.细胞色素P4502C8的基因多态性与用药个体化有着密切的关系.CYP2C8.3是常见的P4502C8的基因多态之一,其发生了双点突变,分别是R139K和K399R.CYP2C8.3主要存在于白种人人群.在本研究里,用大肠杆菌表达了CYP2C8.3和野生型CYP2C8.用UV-Vis,MS和CD光谱表征了纯化的蛋白,考察了它们的热稳定性、底物结合能力、紫杉醇的代谢能力.用stopped-flow研究了野生型CYP2C8和CYP2C8.3代谢紫杉醇过程中的电子传递动力学.结果显示,CYP2C8.3中的突变并没有显著地影响血红素活性位点、蛋白的热稳定性以及紫杉醇的结合能力,但CYP2C8.3的紫杉醇代谢能力却显著地降低到只有野生型的11%.催化过程中,电子从P450还原酶向CYP2C8.3的传递明显慢于P450还原酶向野生型CYP2C8的传递,这可能是CYP2C8.3代谢能力降低的主要原因.CYP2C8.3这个基因多态,在体外显示出对CYP2C8底物极低的代谢能力,可预期其具有重要的临床及病理生理意义,本研究为此方面提供有意义的信息.The cytochrome P450 （CYP） superfamily plays a key role in the oxidative metabolism of a wide range of exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver, and carries out the oxidative metabolism of at least 5% of clinical drugs. Polymorphisms in CYP2C8 have been closely implicated in individualized medication. CYP2C8.3, a common polymorph of CYP2C8 with dual amino acid substitutions R139K and K399R, is found primarily in Caucasians. In this study, CYP2C8.3 and its wild type （WT） CYP2C8 were expressed in E. coli, and their purified proteins were characterized by UV-visible spectroscopy, mass spectrometry, and circular dichroism. Their thermal stability, substrate binding ability, and metabolic activity against paclitaxel were investigated. The electron transfer kinetics during paclitaxel metabolism by WT CYP2C8 or CYP2C8.3 were studied by stopped-flow kinetics. The results revealed that mutations in CYP2C8.3 did not greatly influence the heme active site or protein thermal stability and paclitaxel binding ability, but the metabolic activity against paclitaxel was significantly depressed to just 11% of that of WT CYP2C8. Electron transfer from CYP reductase to CYP2C8.3 was found to be significantly slower than that to WT CYP2C8 during catalysis, and this might be the main reason for the depressed metabolic activity. Since the polymorph CYP2C8.3 is defective in catalyzing substrates of CYP2C8 in vitro, it might be expected to have important clinical and pathophysiological consequences in homozygous individuals, and this study provides valuable information in this aspect.

关 键 词：基因多态 CYP2C8 CYP2C8.3 药物代谢 用药个体化 

分 类 号：Q75[生物学—分子生物学]