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首页> 外文期刊>Biochemistry >Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: Implications for allosteric modulation of cytochrome P450 catalysis
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Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: Implications for allosteric modulation of cytochrome P450 catalysis

机译：基于强机理的9-乙炔基菲对细胞色素P450 2B4的灭活：对细胞色素P450催化的变构调节的意义

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摘要

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k_(inact) are 0.2 and 0.25 min~(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S_(50) of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F′ helix, β1-β2 loop, and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.
机译：研究了基于机制的9-乙炔基菲（9EP）灭活细胞色素P450 2B4（CYP2B4）。分配比和k_（inact）分别为0.2和0.25 min〜（-1）。有趣的是，这种失活表现出乙状动力学，希尔系数为2.5，S_（50）为4.5μM，表明各向同性。酶失活导致apo-CYP2B4的质量增加了218 Da，这是通过电喷雾电离液相色谱法和质谱法确定的，与共价蛋白修饰一致。将经修饰的CYP2B4纯化至均质，并通过X射线晶体学确定其结构。结构表明9EP通过酯键共价结合到Thr 302的Oγ上，这与蛋白质的质量增加是一致的。庞大的菲环的存在导致Phe 297和Phe 206向内旋转，从而导致致密的活性位点。因此，禁止在活性位点结合另一种9EP分子。然而，未经修饰或经9EP修饰的CYP2B4淬灭9EP荧光的结果显示，至少有两个具有不同亲和力的结合位点，低亲和力位点是蛋白质外围的催化位点和高亲和力位点。计算机辅助的对接和分子动力学模拟（结合一个或两个配体）表明，高亲和力位点位于被F'螺旋，β1-β2环和β4环包围的底物进入通道的入口，并起着变构位点以增强9EP炔基的激活效率以及随后对Thr 302的共价修饰。

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