Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4-CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex
Polymorphic Variants of Cytochrome P450 2B6 (CYP2B6.4-CYP2B6.9) Exhibit Altered Rates of Metabolism for Bupropion and Efavirenz: A Charge-Reversal Mutation in the K139E Variant (CYP2B6.8) Impairs Formation of a Functional Cytochrome P450-Reductase Complex
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DOI：
10.1124/jpet.111.183111
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发表时间：
2011-09-01
影响因子：
3.5
通讯作者：
Hollenberg, Paul F.
中科院分区：
文献类型：
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作者：
Zhang, Haoming;Sridar, Chitra;Hollenberg, Paul F.
在本研究中，在重建系统中研究了 CYP2B6 野生型 (CYP2B6.1) 和六种多态性变体（CYP2B6.4 至 CYP2B6.9）对安非他酮、依非韦伦和 7-乙氧基-4-三氟甲基香豆素 (7-EFC) 的代谢，以更好地了解突变对这些天然存在变体的催化特性的影响。所有六种变体均在大肠杆菌中成功过表达，包括 CYP2B6.8（K139E 变体），该变体以前无法在哺乳动物 COS-1 细胞中过表达（J Pharmacol Exp Ther 311：34-43，2004）。安非他酮和依非韦伦的羟基化以及 7-EEC 的 O-脱乙基化的稳态周转率表明这些突变显着改变了 CYP2B6 的催化活性。研究发现，CYP2B6.6 对安非他酮和依非韦伦的羟基化 Km 值分别增加了 4 倍和 27 倍，而 CYP2B6.8 在正常周转条件下完全失去了代谢任何底物的能力。然而，与CYP2B6.1相比，CYP2B6.8在叔丁基过氧化氢作为替代氧化剂存在的情况下保留了77%的7-EEC O-脱乙基酶活性，表明血红素和活性位点具有催化能力。使用停流分光光度法对从 NADPH 依赖性细胞色素 P450 还原酶 (CPR) 到 CYP2B6.8 的电子转移速率进行预稳态测量，结果表明 CYP2B6.8 无法接受来自 CPR 的电子。这些观察结果提供了确凿的证据，表明 K1 39E 变体中的电荷反转突变可阻止 CYP2B6.8 与 CPR 形成功能复合物。这项工作的结果为更好地理解 CYP2B6 多态性和药物代谢的基因型表型相关性提供了进一步的见解。
In this study, metabolism of bupropion, efavirenz, and 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) by CYP2B6 wild type (CYP2B6.1) and six polymorphic variants (CYP2B6.4 to CYP2B6.9) was investigated in a reconstituted system to gain a better understanding of the effects of the mutations on the catalytic properties of these naturally occurring variants. All six variants were successfully overexpressed in Escherichia coli, including CYP2B6.8 (the K139E variant), which previously could not be overexpressed in mammalian COS-1 cells (J Pharmacol Exp Ther 311:34-43, 2004). The steady-state turnover rates for the hydroxylation of bupropion and efavirenz and the O-deethylation of 7-EEC showed that these mutations significantly alter the catalytic activities of CYP2B6. It was found that CYP2B6.6 exhibits 4- and 27-fold increases in the Km values for the hydroxylation of bupropion and efavirenz, respectively, and CYP2B6.8 completely loses its ability to metabolize any of the substrates under normal turnover conditions. However, compared with CYP2B6.1, CYP2B6.8 retains 77% of its 7-EEC O-deethylase activity in the presence of tert-butyl hydroperoxide as an alternative oxidant, indicating that the heme and the active site are catalytically competent. Presteady-state measurements of the rate of electron transfer from NADPH-dependent cytochrome P450 reductase (CPR) to CYP2B6.8 using stopped-flow spectrophotometry revealed that CYP2B6.8 is incapable of accepting electrons from CPR. These observations provide conclusive evidence suggesting that the charge-reversal mutation in the K1 39E variant prevents CYP2B6.8 from forming a functional complex with CPR. Results from this work provide further insights to better understand the genotype phenotype correlation regarding CYP2B6 polymorphisms and drug metabolism.