How do substrates enter and products exit the buried active site of cytochrome P450cam?: 1.: Random expulsion molecular dynamics investigation of ligand access channels and mechanisms
How do substrates enter and products exit the buried active site of cytochrome P450cam?: 1.: Random expulsion molecular dynamics investigation of ligand access channels and mechanisms
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DOI：
10.1006/jmbi.2000.4154
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发表时间：
2000-11-10
影响因子：
5.6
通讯作者：
Wade, RC
中科院分区：
文献类型：
--
作者：
L端demann, SK;Lounnas, V;Wade, RC
细胞色素 P450 形成一个普遍存在的蛋白质家族，其功能包括许多生理上重要的化合物的合成和降解以及外源物质的降解。来自恶臭假单胞菌的细胞色素 P450cam 为理解细胞色素 P450 的结构提供了范例。然而，樟脑（细胞色素 P450cam 的天然底物）访问埋藏活性位点的机制是一个长期存在的谜团。虽然最近有晶体学和模拟证据表明细胞色素 P450BM-3 中的底物进入通道打开，但对于细胞色素 P450cam，在不同的晶体结构中或通过标准分子动力学模拟都没有观察到这种构象变化。在这里，提出了一种新颖的模拟方法，即随机排出分子动力学，其中除了标准分子动力学力场之外，还通过在基板上施加人工随机定向力来找到埋藏活性位点的基板出口通道。随机排斥分子动力学方法通过模拟细胞色素P450BM-3的底物结合结构进行测试，然后应用于细胞色素P450cam与不同底物和产物的复合物。确定了三种途径，其中一种对应于先前基于晶体学和定点诱变数据提出的通道。没有观察到通过之前被认为是产品出口通道的充满水的通道的出口。通过随机排斥分子动力学方法获得的路径与通过晶体学 B 因子分析获得的热运动路径非常匹配。与在细胞色素 P450BM-3 中观察到的用于棕榈油酸退出的大骨架运动（高达 4 埃）相反，樟脑通过细胞色素 P450cam 仅需要小骨架运动（小于 2.4 埃）与侧链旋转相结合。与此同时，在几乎所有的退出轨迹中，被认为充当蛋白质二级结构元件之间的离子系链的盐键都受到干扰。 (C) 2000 年学术出版社。
Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. in contrast to large backbone motions (up to 4 Angstrom) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 Angstrom) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed. (C) 2000 Academic Press.