【作者】 李雪；

【导师】 张红星；

【作者基本信息】 吉林大学 ， 物理化学， 2012， 博士

【摘要】 酶是由活细胞产生的具有催化功能的蛋白质。它属于生物大分子，但只有很小的部分起到结合底物或催化反应的作用，称为酶的活性中心。在酶催化过程中，底物或底物类似物在酶的活性中心瞬间生成酶-底物复合物，这个复合物具有较高的能量，是一种极为不稳定的状态。这种瞬间的状态很难通过实验方法来捕获。计算机模拟的出现有力的弥补了实验上的不足，能够从原子水平上获得整个反应历程的详细信息。通过分析酶催化反应的中间体和过渡态的结构，并结合动力学方法确定反应的限速步骤，就能够推知整个反应的催化机理。本文主要利用量子化学计算方法，基于从蛋白质晶体结构中截取的团簇模型，对N~5-羧基氨基咪唑核苷酸变位酶、α－甲酰基辅酶A消旋酶和谷胱甘肽硫转移酶这三个体系进行了催化机理的理论研究。主要结果如下：1.N~5-羧基氨基咪唑核苷酸变位酶催化的重排反应对于除原生动物外的所有生命体来说，从头合成嘌呤这一过程都至关重要。在Class I PurE催化下生成5-氨基-4-羧基咪唑核苷酸(CAIR)这一反应是整个嘌呤合成过程中的关键步骤。遗传研究表明，Class I PurE对微生物的生长必不可少。这种酶的缺失会导致嘌呤缺陷体，在人类和鼠的血清中，这种缺陷体不具备繁殖能力，也没有抵御疾病的能力。本文利用密度泛函理论B3LYP方法对N~5-羧基氨基咪唑核苷酸变位酶催化的重排反应机理进行了理论研究。基于EcPuE的晶体结构，我们构建了包含93个原子的理论模型来研究其催化机理，并利用自然键轨道理论(NBO)方法进行了电荷布居分析，进一步验证推测机理的正确性。结果表明，此重排反应分脱羧和羧化两个步骤进行。His45是一个既能起到广义酸作用又能起到广义碱作用的催化残基。在计算过程中，我们得到一个具有四面体结构的中间体（iso.CAIR），从理论上证实了之前实验上对该结构的预测。根据对每个优化结构的能量计算，绘制出总反应的势能面曲线，通过比较，AIR的羧化反应步骤为整个反应的限速步骤。同时，我们也考察了其它重要氨基酸残基（Ser43,Ala44, His45, Gly71, His75和Leu76等）对催化反应的影响。2.α－甲酰基辅酶A消旋酶催化机理的理论研究支链脂肪酸是人类日常饮食中的重要成分，也被用于非类固醇抗炎药物，如布洛芬。在代谢过程中，只有S构型底物能够通过支链脂酰辅酶A氧化酶发生代谢作用。α－甲酰基辅酶A消旋酶(AMACR)能够使各种α-甲酰基辅酶A衍生物发生S、R异构互变。因此，AMACR的这一功能使其成为必需酶。研究发现它在乳腺癌、直肠癌和卵巢癌中存在过表达现象。此外，AMACR是前列腺癌的高度敏感及特异的标志物，在前列腺癌的早期诊断中具有十分重要的临床应用价值。在本文中，我们对来源于结核分枝杆菌的α－甲酰基辅酶A消旋酶(MCR)进行了深入研究。它与人类的氨基酸序列有着高达43%的同源性。计算选用B3LYP方法，结构的优化和单点能的计算分别在6-31G(d)和6-311++G(2d,2p)两个基组水平上完成。计算结果阐明了MCR催化反应的机理，支持Prasenjit Bhaumik等人通过实验方法预测的结果。在1,1-质子转移反应中，His126和Asp156作为广义碱和广义酸催化反应的进行。从优化的结构中可以看出，去质子反应后产生的中间体为平面烯醇负离子式，因此烯醇式或烯酮式的中间体构型被排除。通过对能垒的比较，我们认为在从S-异构体向R-异构体转变的过程中，去质子化步骤是整个消旋反应的限速步骤。应用理论方法详细地描述酶促反应的路径，将对基于过渡态结构设计和研发抗癌药物提供有力的帮助。3.谷胱甘肽（GSH）激活机理的理论研究谷胱甘肽硫转移酶（GSTs）能够引起亲电类毒性物质的代谢，因此谷胱甘肽硫转移酶具有重要的解毒作用。GSTs是通过催化谷胱甘肽（GSH）与一系列亲电类的毒性物质发生反应，生成无毒的可溶性物质，之后被排除细胞外以达到解毒目的。但是，它对药物的代谢会产生抗药性问题，导致临床治疗的失败。GSH在与亲电试剂反应之前首先要被激活为硫醇离子的形式。关于典型的GSTs—GSTA1-1, GSTP1-1和GSTM1-1中GSH的激活机理已经有了广泛的研究。而非典型的GSTs—Gtt2的激活机理研究尚未见报道。本文应用Gaussian 03程序，采用B3LYP方法完成所有的几何构型优化、频率分析和能量计算，推测出新型谷胱甘肽硫转移酶Gtt2中谷胱甘肽（GSH）的激活机理。本文提出的GSH激活机理与实验假设相一致,并且从能量角度证明了该机理的合理性。在反应过程中，His133作为广义碱、水分子作为助催化剂。通过与典型GSTs的比较，分析了产物具有较高能量的原因。我们在计算过程中选取了两个模型，分别含有不同数量的水分子，考察了体系中水分子对GSH激活机理的影响。更多还原

【Abstract】 Enzymes are catalysts that produced by living cells. Virtually all of themare protein. Enzymes belong to the biological macromolecule, but the regionthat binds the substrate and converts it into product is a relatively small part.This region is known as the active center. The enzyme combines with itssubstrate to form an enzyme-substrate complex. It is an extremely unstablestate and has the highest free energy in the reaction pathway. This transientspecie is difficult to capture by the experimental techniques. Computersimulation can make up the shortfall and give the detailed information of thecatalytic reaction on the atomic level. By analysis of the intermediates andtransition states, and binding the kinetic method to determine the rate-limitingstep, we can infer the catalytic mechanism of the enzymes.1. The mechanism of rearrangement reaction catalyzed byN5-carboxyaminoimidazole ribonucleotide mutaseIn this paper, quantum chemistry calculation methods were used. Thecluster model for the simulations was constructed on the basis of crystal structure. We have studied the catalytic mechanism ofN5-carboxyaminoimidazole ribonucleotide mutase, glutathione transferaseGtt2 andα-Methylacyl-CoA racemase using theoretical method. The mainresults are summarized as follows:De novo purine biosynthesis is central to all life forms except protozoa.In this process, the carboxylationof 5-aminoimidazole ribonucleotide (AIR) isconverted to 4-carboxyaminoimidazole ribonucleotide (CAIR) catalyzed byClass I PurE. The necessity of Class I PurE for the growth of microbe hasbeen shown by genetic studies. Deletion of this enzyme will result in a purineauxotroph that is unable to propagate in human or mouse serum and is notviable in animal models predictive of disease.In the present study, the complete reaction mechanism of PurE has beeninvestigated by using the density functional theory with the hybrid B3LYPfunctional. The model containing 93 atoms was constructed on the basis ofthe X-ray structure of PurE from Escherichia coli. Additionally, we hadpreceded the charge population analysis with the method of the Natural BondOrbital (NBO) to verify the soundness of the proposed mechanism. Theresults show that this rearrangement reaction is carried out in two steps, one isdecarboxylation and the other is carboxylation. The conserved residue His45plays an essential catalytic role as both general aicd and general base. Atetrahedral intermediate iso.CAIR is observed during the carboxylationreaction and it is a plausible construction proved in experiment. The potential energy curves are drawn in terms of single-point energies. By comparing theenergy barriers, we could consider the carboxylation step as the rate-limitingstep. Furthermore, other residues including Ser43, Ala44, His45, Gly71,His75 and Leu76 also play important roles via the strong hydrogen-bondinteractions.2. Reaction mechanism ofα-Methylacyl-CoA racemase: Atheoretical investigationThe branched-chain fatty acids are important component in the humandiet. Also, they are used for non-steroidal anti-inflammatory drugs such asibuprofen. In their metabolic process, only the (S)-substrate can bemetabolized through the branched-chain acyl-coA oxidases.α-Methylacyl-CoA racemase (AMACR) catalyzes the conversion fromR-enantiomer to S-enantiomer at the 2-position of fatty acyl-CoA esters.Therefore, AMACR is the essential enzyme in the metabolic process. Inaddition, AMACR can be used as highly sensitive and specific marker forprostate cancer. It has very important clinical value in the early diagnosis ofprostate cancer. Subsequently, the over-expression has also been found inbreast, colorectal and ovarian cancer.In this paper, amethylacyl-CoA racemase from Mycobacteriumtuberculosis (MCR) were studied. The amino acid sequence identity betweenMCR and human is up to 43%. The calculation was completed by using thedensity functional theory (DFT) B3LYP method. The geometry optimizations were done with 6-31G (d) basis set and the single-point energy calculationswere done with 6-311 + + G (2d, 2p) basis set. Our calculation resultsillustrate the catalytic mechanism of MCR, and support the experimentalresults proposed by Prasenjit Bhaumik. His126 and Asp156 function as thegeneral base and the general acid respectively in the 1, 1 - proton transferreaction. It can be seen from the optimized structure, the intermediate is aplane enolate anion type, so the type of the enol or ketene is excluded. Bycomparing the energy barrier, we consider that the conversion from theS-enantiomer to the R- enantiomer is the rate-limiting step during theracemization. The theoretical studies on the mechanism of 1, 1 - protontransfer reaction catalyzed by MCR will be helpful to synthetize theanti-cancer drugs which designed based on the transition state structure.3．The theoretical study on the GSH activation mechanismGlutathione S-transferase (GSTs) can cause the metabolism ofelectrophilic toxic substances. So it has an important role in detoxification.Glutathione (GSH) can react with a range of electrophilic toxic substances,and generate the non-toxic soluble substances, then the products are excludedfrom the cell. However, the drug metabolism will result in drug resistance,even the failure of clinical treatment. Before it reacting with the electrophilicsubstrates, GSH should be activated into the thiolate form. Recently, the GSHactivation mechanism catalyzed by typical GSTs (GSTA1-1, GSTP1-1 andGSTM1-1) has been proposed. How about the GSH activation mechanism catalyzed by the atypical GST (Gtt2)?In the present study, all geometry optimizations, single-point calculationsand frequency analysis were performed using density functional theory (DFT)with the B3LYP functional and the basis set 6-31G (d) as implemented inGaussian03. The GSH activation mechanism of Gtt2 has been investigatedand the results are consistent with the experimental data available. The protontransfer mediated by a water molecule is energetically feasible. Theimportance of water molecule as promoter is unequivocal. His133 functionsas general base in the reaction. The reason why the product has a higherenergy has been analyzed by comparing with the typical GSTs. In addition,another model that including a smaller amount of water molecules wasselected. The effects of water molecules in the system were investigated. Itproved that different model does not cause the changes of the pathway, but theenergy and configuration will be affected.更多还原