三种生物降解酶催化机理的理论研究

发布时间：2018-05-12 07:02

本文选题：酶催化 + 反应机理　；参考：《山东大学》2016年硕士论文

【摘要】：酶催化反应因具有催化效率高、反应条件温和等优点,在日常生活和工农业业生产中被广泛应用。然而,由于酶促反应体系的复杂性,目前从实验上检测反应过程中的中间体和过渡态的结构还十分困难,对酶反应机理的认知主要是根据动力学和突变试验来推测。随着量子化学理论方法和计算机技术的不断发展,以量子化学为基础的理论与计算化学可以从原子水平上研究化学反应的过程,给出酶催化反应的详细机理、能量关系及决速步骤等信息,在研究生物大分子反应机理方面具有突出优势。本论文以最新测得的三种生物降解酶的晶体结构为基础,运用量子力学与分子力学(QM/MM)相结合的方法对这些酶的催化机理进行了较为系统的理论研究,在原子水平上给出了反应的细节,包括过渡态、中间体的构型、能量关系、决速步骤等,阐明了一些活性残基在催化中的不同作用,并对部分残基进行了突变计算,研究结果不仅弥补了实验研究的不足,而且为相关酶的实验研究及应用开发提供了必要的基础。主要研究内容包括：(1)N-酰基高丝氨酸内酯酶催化机理的理论研究自然界中,许多致病的细菌通过群体感应效应使动植物以及人类患病。N-酰基高丝氨酸内酯(N-acyl-homoserine lactones, AHLs)作为一种群体感应信号分子在革兰氏阴性细菌中被广泛利用。N-酰基高丝氨酸内酯酶通过降解AHLs分子,来阻止群体感应发生,从而起到预防疾病的作用。实验上建议反应过程内酯环以分步的方式断裂。本文利用QM/MM方法研究了N-酰基高丝氨酸内酯酶的催化机理,计算结果表明反应过程中的开环反应是一个协同反应而不是分步反应。通过研究MM区中重要残基的静电作用,确定了周围残基对催化反应的影响,为进一步进行突变实验提供了理论指导。我们的研究对实验上提出的反应机理进行了部分修改,确定了详细的反应机理及其能量学信息,有助于理解高丝氨酸类内酯酶的降解机理,为治疗因高丝氨酸信号分子引起的疾病提供了理论指导。(2)2-吡喃酮-4,6-二羧酸水解酶催化机理的理论研究2-吡喃酮-4,6-二羧酸(PDC)是木质素降解过程中的一种中间体。木质素的降解对自然界中碳循环具有重要意义。2-吡喃酮-4,6-二羧酸(PDC)水解酶催化PDC水解生成两种水解产物,分别为烯醇式产物4-羧基-2-羟基粘康酸(CHM)和酮式产物4-草酰中康酸(OMA)。本文利用QM/MM方法研究了PDC水解酶的催化机理。根据我们的计算结果可得到如下信息：整个水解反应包括三个基元反应,由于第三步(氢转移)反应有两种可能的途径,因而可以得到两种水解产物；通过突变研究,确定了三个组氨酸(His31、His33和His180)在反应过程中的作用；反应过程中反应物、过渡态和中间体的构型；整个反应过程的势能面。本论文对研究PDC聚合物的生物降解具有理论指导意义。(3)2,4'-二羟基苯乙酮加双氧酶催化机理的理论研究2,4'-二羟基苯乙酮(DHAP)是木质素的一种降解产物,2,4'-二羟基苯乙酮加双氧酶(DAD)可以把DHAP进一步降解成对羟基苯甲酸和甲酸。对于DAD的催化机理,实验学家通过研究该酶的仿生模型给出了一条涉及一个四元环中间体的生成与断裂的机理。本文利用QM/MM方法研究了2,4'-二羟基苯乙酮加双氧酶的催化机理。根据我们的计算结果涉及四元环的路径能垒过高,于是我们对其他可能的机理进行了研究。结果表明,DAD采用一种新的反应机理催化2,4'-二羟基苯乙酮的降解,总反应包含八步基元反应,其中前两步机理与实验上建议的机理相同,最后一步基元反应是决速步。因为三重态的氧气与五重态的铁作用可得到三种可能的自旋多重度(三重态、五重态和七重态),所以我们对一些关键步骤分别在三重态、五重态和七重态条件下进行了计算。结果表明,反应首先经历一个从三重态到五重态的转变,随后反应主要在五重态势能面上进行。计算结果为研究金属酶催化的脂肪族类碳碳键的断裂反应提供了理论指导。
[Abstract]:The enzyme catalyzed reaction has been widely used in daily life and industrial and agricultural production because of its high catalytic efficiency and mild reaction conditions. However, because of the complexity of the enzymatic reaction system, it is difficult to detect the structure of intermediate and transition state in the reaction process at present, and the cognition of the mechanism of the enzyme reaction is mainly based on the fact that the enzyme catalyzed reaction system is complex. With the continuous development of quantum chemical theory and computer technology, the theoretical and computational chemistry based on quantum chemistry and computational chemistry can study the process of chemical reactions from the atomic level, give information on the detailed mechanism, energy relations and the rate of determination of the enzyme in the study of biological macromolecules. Based on the crystal structure of the latest three biodegradable enzymes, this paper systematically studies the catalytic mechanism of these enzymes by combining the method of quantum mechanics and molecular mechanics (QM/MM), and gives the details of the reaction at the atomic level, including the transition state and the intermediate. The structure, the energy relation, the quick step and so on, clarify the different effects of some active residues in the catalysis, and calculate the mutation of some residues. The results not only make up the shortage of the experimental research, but also provide the necessary foundation for the experimental research and application development of the related enzymes. The main contents include: (1) the high acyl group of N- The theoretical study of the catalytic mechanism of the serine lactone enzyme in nature, many pathogenic bacteria are widely used as a kind of N-acyl-homoserine lactones (AHLs), the.N- acyl high serine lactone (lactones, AHLs), in gram - acyl high serine lactone in gram - negative bacteria through the quorum induction effect By degrading AHLs molecules to prevent the occurrence of quorum induction, the enzyme plays a role in preventing disease. In the experiment, it is suggested that the ester ring breaks in a stepwise way. In this paper, the catalytic mechanism of N- acyl hserine lactone enzyme was studied by QM/MM method. The results show that the ring opening reaction in the reaction process is a synergistic reaction. It is not a step reaction. Through the study of the electrostatic action of the important residues in the MM region, the influence of the surrounding residues on the catalytic reaction is determined, and the theoretical guidance is provided for the further mutation experiment. Our research has made some modifications to the reaction mechanism proposed in the experiment, determined the detailed reaction mechanism and the energy information, which is helpful to the theory of the reaction. The degradation mechanism of high serine lactone enzyme can provide theoretical guidance for the treatment of diseases caused by high serine signal molecules. (2) theoretical study on the catalytic mechanism of 2- -4,6- two carboxylic acid hydrolase, 2- (PDC) is an intermediate in the degradation of lignin. The degradation of lignin in natural carbon cycle The.2- hydrolysis of pyruvone -4,6- two carboxylic acid (PDC) hydrolase catalyzes the hydrolysis of PDC to produce two kinds of hydrolysates, which are 4- carboxylic -2- hydroxyviscoacic acid (CHM) and 4- oxacylic acid (OMA) in the enol product, respectively. This paper studies the catalytic mechanism of PDC hydrolase by QM/MM method. The following information can be obtained according to our calculation results. The whole hydrolysis reaction includes three elemental reactions, and because there are two possible pathways in the third step (hydrogen transfer) reaction, two kinds of hydrolysates can be obtained. Through the mutation study, the role of three histidine (His31, His33 and His180) in the reaction process is determined; the configuration of the reactant, transition state and intermediate in the reaction process; The potential energy surface of the reaction process. This paper has theoretical guiding significance for the study of biodegradation of PDC polymers. (3) theoretical study on the catalytic mechanism of 2,4'- two hydroxybenzophenone plus dioxygen enzyme, 2,4'- two hydroxyacetophenone (DHAP) is a degradation product of lignin, and 2,4'- two hydroxyacetophenone plus dioxygen enzyme (DAD) can further degrade DHAP into DHAP In the catalytic mechanism of hydroxy benzoic acid and formic acid. By studying the bionic model of the enzyme, an experimenter gives a mechanism involving the formation and fracture of a four membered ring intermediate. In this paper, the catalytic mechanism of 2,4'- two hydroxyacetophenone plus dioxygenase is studied by QM/MM method. According to our calculation, the four membered ring is involved. The path energy barrier is too high, so we have studied other possible mechanisms. The results show that DAD uses a new reaction mechanism to catalyze the degradation of 2,4'- two hydroxy acetophenone. The total reaction contains eight steps of the basic reaction, of which the first two steps are the same as that suggested in the experiment, and the final step reaction is a quick step. Because of the three heavy state. The action of oxygen and iron in the five state can be obtained by three possible spin multiples (three, five, and seven heavy). So we calculate some of the key steps in three, five and seven states, respectively. The results show that the reaction first experienced a transition from three to five, then the reaction was mainly in five. The calculated results provide theoretical guidance for the study of the fracture reaction of fatty carbon carbon bonds catalyzed by metalloenzymes.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O629.8

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