CYP2E1 hydroxylation of aniline involves negative cooperativity
CYP2E1 hydroxylation of aniline involves negative cooperativity
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DOI：
10.1016/j.bcp.2013.12.003
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发表时间：
2014-02-01
影响因子：
5.8
通讯作者：
Miller, Grover P.
中科院分区：
文献类型：
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作者：
Hartman, Jessica H.;Knott, Katie;Miller, Grover P.
CYP 2 E1在苯胺的代谢活化和消除中发挥作用，但关于其作用机制的报告相互矛盾，因此与苯胺代谢相关。基于我们对类似化合物的研究，我们假设苯胺在代谢过程中结合两个CYP 2 E1位点，导致协同反应动力学，并通过严格的体外研究验证了这一假设。基于Hill方程的数据拟合（n = 0.56），重组CYP 2 E1的动力学特征显示出显著的负协同性。从机制上讲，数据得到了最好的解释，通过一个两个结合位点的合作模型，苯胺结合高亲和力（KS =30 μ M），其次是第二个较弱的结合事件（KS = 1100 μ M），导致在氧化速率的三倍增加。通过4-甲基吡唑的抑制研究证实了苯胺的结合位点。人肝微粒体抑制剂表型实验验证了CYP 2 E1在苯胺羟基化中的核心作用，并表明CYP 2A 6和CYP 2C 9的次要作用。重要的是，次要代谢途径的抑制导致微粒体CYP 2 E1的动力学特征，其复制了重组酶的首选机制和观察到的参数。苯胺的体外CYP 2 E1代谢至体内清除率的规模化建模，尤其是在低苯胺水平下，导致与基于非合作性Michaelis Menten动力学的传统模型存在显著偏差。这些发现提供了关于CYP 2 E1在苯胺代谢活化和消除中的潜在重要性的关键机制观点，以及CYP 2 E1催化的负协同代谢反应的第一个实验证据。(C)2013 Elsevier Inc. All rights reserved.
CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n = 0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (K-s =30 mu M) followed by a second weaker binding event (K-ss = 1100 uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1. (C) 2013 Elsevier Inc. All rights reserved.