细胞色素酶CYP2E1与芳香族底物结合的生物信息与结构生物学研究

发布时间：2018-08-11 17:26

【摘要】：细胞色素酶P450家族是和人体内药物代谢关系最为密切的一类药物代谢酶大家族。CYP2E1,作为细胞色素酶家族的一员,负责其中6%的药物代谢,并且其代谢的药物种类相当广泛,有着很不同的结构和化学性质,比如,酒精类,单环小分子,两元杂环小分子,甚至是脂肪酸。然而,最近展示的CYP2E1晶体结构表明,它有着细胞色素酶家族中最小的活性口袋容积,另外除了通往活性位点的离子通道,它附近还存在着另一个小型的空间,这在细胞色素酶家族中十分罕见。作为这么一个有限的空间,它是如何做到代谢这么多不同性质的底物的,这是个很值得研究的问题。不仅如此,CYP2E1针对单环小分子底物也在一些代谢反应时显示了反常的动力学曲线,这显示了两个小分子可能同时结合在酶上的负协同效应,引起代谢抑制。现在关于CYP2E1的底物代谢多样性和其负协同效应的研究并不十分透彻。所以本着进一步了解CYP2E1活性位点的特性的目的,我们选取五个不同结构、性质的芳香环小分子分子(苯,苯胺,对乙酰氨基酚,氯唑沙宗和茶碱)对接进入CYP2E1的晶体结构,接着进行5ns的分子动力学模拟,来研究它在人体内的底物代谢多样性的机理。同时,为了进一步了解负协同效应的机制,苯胺作为有着最小取代基的芳香单环底物,被挑选出将一个以及两个小分子对接入CYP2E1蛋白质,并进行10ns的分子动力学模拟。得到结果之后我们又进行了很细致的均方差位移,近距离相互作用分析。对于探索负协同效应部分,我们又进行了协相关度的分析和吉布斯结合自由能的计算,得到了很有价值的成果。模拟展示了芳香小分子和CYP2E1的活性位点相互作用主要通过和Phe106,Phe116,Phe207,Phe298以及Phe478的π-π堆积芳香相互作用,几乎没有氢键作用。并且不同的小分子蛋白复合物根据结合能产生的构象聚类特征和分子动力学模拟中由于分子运动所产生的结构特征,在决定代谢产物多样性方面起了重要作用。单产物的小分子通常只具有一个基于能量的构象聚类结果,比如苯和氯唑沙宗。而产物越多,其聚类结果越多样,动力模拟的活动越活跃,比如对乙酰氨基酚,有着四个可能的代谢位点,甚至在模拟过程中由于活动性太高,进入了活性口袋附近的小空间内。另外,CYP2E1的负协同作用来源于第二个单环小分子通过和Phe478,连接离子通道和活性位点的最重要的残基,产生π-π堆叠,并加强活性位点的小分子同样和Phe478产生的偏移平行π-π堆叠,产生能量稳定的三叠体π-π堆叠,限制并扰乱了待反应小分子的正常活动。第二个小分子的进入也降低了在小分子定位上最关键的Thr303的活性。所有这些发现都为CYP2E1的底物代谢多样性和负协同效应的机理研究提供了原子角度的分析,为将来需要的个性化药物的新发展做贡献。
[Abstract]:The cytochrome P450 family is one of the most closely related drug metabolizing enzymes in human body. As a member of the cytochrome enzyme family, it is responsible for 6% of the drug metabolism. They have very different structures and chemical properties, such as alcohol, monocyclic, binary heterocyclic, and even fatty acids. However, the recently shown crystal structure of CYP2E1 suggests that it has the smallest active pocket volume in the cytochrome family, and that in addition to the ion channel leading to the active site, there is another small space nearby. This is rare in the cytochrome family. As such a limited space, it is worth studying how it metabolizes so many different substrates. Moreover, CYP2E1 also showed abnormal kinetic curves in some metabolic reactions against monocyclic small molecular substrates, which indicated that two small molecules may bind to negative synergistic effect on enzyme at the same time, resulting in metabolic inhibition. Current studies on the metabolic diversity of substrates and their negative synergistic effects of CYP2E1 are not very thorough. Therefore, for the purpose of further understanding the properties of CYP2E1 active sites, we selected five aromatic ring molecules (benzene, aniline, paracetamol, chlorzoxazol Zong He theophylline) with different structures to dock into the crystal structure of CYP2E1. Then the molecular dynamics simulation of 5ns was carried out to study the mechanism of its metabolic diversity in human body. At the same time, in order to further understand the mechanism of negative synergism, aniline, as an aromatic monocyclic substrate with the smallest substituent, was selected to attach one or two small molecules to CYP2E1 protein and to simulate the molecular dynamics of 10ns. After we get the results, we make a very detailed analysis of the RMS displacement and the close-distance interaction. For the exploration of negative synergistic effect, we analyze the degree of cocorrelation and calculate Gibbs' combined free energy, and obtain valuable results. The simulation shows that the interaction between aromatic molecules and active sites of CYP2E1 is mainly through the interaction with Phe106, Phe116, Phe207, Phe298 and Phe478 蟺-蟺 piling aromatics. There is almost no hydrogen bond interaction between the aromatic molecules and the active sites of Phe106, Phe116, Phe207, Phe298 and Phe478. And different small molecular protein complexes play an important role in determining the diversity of metabolites according to the conformational clustering characteristics of binding energy and the structural characteristics of molecular motion in molecular dynamics simulation. Single-product small molecules usually have only one energy-based conformation clustering result such as benzene and chlorzoxazone. And the more products, the more diverse the clustering results, the more dynamic simulation activities, such as acetaminophen, has four possible metabolic sites, even in the simulation process because of high activity, Into a small space near the active pocket. In addition, the negative synergistic effect of CYP2E1 comes from the second monocyclic small molecule passing through Phe478, the most important remnant of ion channel and active site, to produce 蟺-蟺 stacking, and to enhance the stacking of small molecules at active sites in parallel with the shift 蟺-蟺 produced by Phe478. The formation of an energy-stable triplet 蟺-蟺 stacks limits and disrupts the normal activity of small molecules to be reacted. The entry of the second small molecule also reduced the activity of Thr303, which is the most critical for the localization of small molecules. All these findings provide atomic angle analysis for the study of the mechanism of substrates metabolic diversity and negative synergism of CYP2E1 and contribute to the new development of individualized drugs needed in the future.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341

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