【作者】 周奕含；

【导师】 唐敖庆； 李泽生；

【作者基本信息】 吉林大学 ， 物理化学， 2008， 博士

【摘要】 本文利用分子力学和分子动力学方法,对三种酶的体系进行了三维结构预测及分子对接的理论研究。主要内容包括:1.利用分子动力学模拟建立了无底物的人类细胞色素CYP2C9及其变体CYP2C9*13的三维结构,确定了CYP2C9*13中Pro与Asp之间的肽键为反式构型,并从结构上解释了CYP2C9*13催化活性降低的原因。在此基础上进行了两种底物药的对接研究,与动力学实验结果符合得很好。进一步通过分子动力学模拟建立了六个90位突变的变体酶的三维结构,其活性口袋入口及底物与酶的相互作用,以及蛋白的稳定性的改变,体现了第90位氨基酸在CYP2C9蛋白三维结构中的重要作用。2.利用同源模建和分子动力学模拟方法,模建了大肠杆菌中在蛋氨酸生物合成中起关键作用的高丝氨酸琥珀酰基转移酶的三维结构。分析了活性位点的组成,并通过与其天然底物琥珀酰-辅酶A的对接研究,从理论上揭示EcHTS的催化机理并确认了对复合物形成起到重要作用的氨基酸残基。3.利用同源模建和分子动力学模拟方法,模建了两个新发现的a/β水解酶超家族成员W14和W15的三维结构。分析并比较了w14和W15的活性位点的组成与结构,并通过与旺.醋酸萘酯的对接研究,从理论上提出Gly82和Vall3为形成“氧洞”的关键残基,有利于稳定水解过程中的带负电的过渡态,同时也确认了其他对复合物形成起到重要作用的氨基酸残基。更多还原

【Abstract】 With the development of computer technology, medicine chemistry, molecular biology and computational chemistry,computer aided molecular modeling has become a very important and active research field. In this thesis, molecular mechanics and molecular dynamics methods are used to build three dimensional models for three enzymes and study their structures properties in detail. Some creative results were obtained from the methods mentioned above. The main results are outlined as follows:1. The structures and function research of human CYP2C9 and CYP2C9*13 variant. CYP2C9*13 is a new allele of human CYP2C9 characterized by mutation encoding Leu90Pro substitution. The three-dimensional structure models of the substrate-free CYP2C9*1 and its variant CYP2C9*13 were constructed by molecular dynamics simulations. The trans configuration of the bond between Pro90 and Asp89 in CYP2C9*13 is firstly identified. The structure change caused by Leu90Pro replacement was revealed and used to explain the dramatic decrease of the enzymatic activity. Consequent docking study is consistent with the results of the kinetic experiments.The 3D structures of variants, L90I, L90G, L90R, L90Q and L90W were constructed by molecular dynamics for further research. The results suggest that the changes in catalysis activity by the substitution at residue 90 are due to changes in size of entrance, interaction between substrates and the proteins, and stability. Thus the residue 90 of CYP2C9 would have an important role on the enzyme.2. Homology modeling and docking studies of homoserine transsuccinylase from Escherichia coli. The three dimensional structure of homoserine transsuccinylase from Escherichia coli (EcHTS) was modeled by using homology and molecular dynamics methods. On the basis of the modeling, the components of active site in EcHTS were analyzed. The structure analysis shows that Cys142 but not Lys47 is the main residue for the nucleophile during the catalytic reaction. The docking of succinyl-CoA with EcHTS has also been performed, and the important residues for binding were identified.3. Homology modeling and docking studies of novelα/βhydrolase fold proteins W14 and W15. The three dimensional structure of novelα/βhydrolase fold proteins W14 and W15 were modeled by using homology and molecular dynamics methods. On the basis of the modeling, the components and the structures of active sites in W14 and W15 were analyzed and compared. The docking of 1-naphtyl acetate with the two proteins have also been performed and compared. Gly82 and Val13 were key residues to form the oxyanion-hole and stabilized the negatively charged transition state that occurs during hydrolysis. Other important residues for binding were also identified.更多还原