Structural analysis of mammalian cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene: insight into partial enzymatic activity.
Structural analysis of mammalian cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene: insight into partial enzymatic activity.
复制标题
DOI：
10.1021/bi200482g
复制
发表时间：
2011-06-07
期刊：
影响因子：
2.9
通讯作者：
Halpert JR
中科院分区：
文献类型：
--
作者：
Gay SC;Zhang H;Wilderman PR;Roberts AG;Liu T;Li S;Lin HL;Zhang Q;Woods VL Jr;Stout CD;Hollenberg PF;Halpert JR
对兔细胞色素P450 2B4与基于机理的灭活剂叔丁基苯乙炔(TBPA)共价结合的结构和计算的结合分析揭示了该酶是如何保持部分活性的。由于与tBPA的结合修饰了高度保守的活性中心残基，先前研究中观察到的tBPA标记的2B4的残留活性令人费解。在这里，我们描述了一个修饰的哺乳动物P450的第一个晶体结构，它显示了tBPA的氧化代谢产物与螺旋I的Thr302结合。这些结果与先前确定Thr302为结合位点的研究一致。在每种结构中，2B4的核心保持不变，但塑性区的排列不同。这导致了一个紧凑和封闭的结构。在这种构象中，tBPA指向螺旋B‘，与血红素平面成31°角。这种构象与以前在电学实验中进行的结果是一致的。然而，由于B/C环和螺旋F通过G的移动而引起的另一种结构中的2B4的二聚化改变了tBPA的位置。在这种情况下，tBPA几乎平行于血红素平面，这是因为相反单体的螺旋F‘进入活性位置以稳定二聚体。然而，使用这种开放形式的对接实验表明，tBPA能够向上旋转，使睾酮和7-乙氧基-4-三氟甲基香豆素能够访问血红素，这可以解释之前观察到的部分活性。
A combined structural and computational analysis of rabbit cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene (tBPA) has yielded insight into how the enzyme retains partial activity. Since conjugation to tBPA modifies a highly conserved active site residue, the residual activity of tBPA-labeled 2B4 observed in previous studies was puzzling. Here we describe the first crystal structures of a modified mammalian P450, which show an oxygenated metabolite of tBPA conjugated to Thr 302 of helix I. These results are consistent with previous studies that identified Thr 302 as the site of conjugation. In each structure, the core of 2B4 remains unchanged, but the arrangement of plastic regions differs. This results in one structure that is compact and closed. In this conformation, tBPA points toward helix B′, making a 31° angle with the heme plane. This conformation is in agreement with previously performed in silico experiments. However, dimerization of 2B4 in the other structure, which is caused by movement of the B/C loop and helices F through G, alters the position of tBPA. In this case, tBPA lies almost parallel to the heme plane due to the presence of helix F′ of the opposite monomer entering the active site to stabilize the dimer. However, docking experiments using this open form show that tBPA is able to rotate upward to give testosterone and 7-ethoxy-4-trifluoromethylcoumarin access to the heme, which could explain the previously observed partial activity.
登录
查看更多内容
影响因子：
5.8
作者：
HALPERT, J
通讯作者：
HALPERT, J
DOI：
10.1124/jpet.300.2.549
发表时间：
2002-02-01
影响因子：
3.5
作者：
Kent, UM;Mills, DE;Hollenberg, PF
通讯作者：
Hollenberg, PF
DOI：
10.1124/jpet.109.158782
发表时间：
2009-11-01
影响因子：
3.5
作者：
Lin, Hsia-lien;Zhang, Haoming;Hollenberg, Paul F.
通讯作者：
Hollenberg, Paul F.
影响因子：
15
作者：
Mak, Piotr J.;Zhang, Haoming;Kincaid, James R.
通讯作者：
Kincaid, James R.
DOI：
10.1107/s090744499900846x
发表时间：
1999-10-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Leslie, AGW
通讯作者：
Leslie, AGW