Potent Mechanism-Based Inactivation of Cytochrome P450 2B4 by 9-Ethynylphenanthrene: Implications for Allosteric Modulation of Cytochrome P450 Catalysis

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• 作者：Haoming Zhang ; Sean C. Gay ; Manish Shah ; Maryam Foroozesh ; Jiawang Liu ; Yoichi Osawa ; Qinghai Zhang ; C. David Stout ; James R. Halpert ; Paul F. Hollenberg
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：January 15, 2013
• 年：2013
• 卷：52
• 期：2
• 页码：355-364
• 全文大小：434K
• 年卷期：v.52,no.2(January 15, 2013)
• ISSN：1520-4995

文摘

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^鈥?, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S₅₀ of 4.5 渭M 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 O纬 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鈥?helix, 尾1鈭捨? loop, and 尾4 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.