几类金属酶和糖苷酶催化机理的理论研究

发布时间：2018-04-20 16:06

本文选题：酶催化反应 + 反应机理　；参考：《山东大学》2017年博士论文

【摘要】：酶是一切生命活动的基础,几乎所有细胞内的代谢反应都离不开酶的参与。正是由于酶的催化作用,生物代谢活动中产生的物质和能量才能满足生物体的需求。由于酶具有传统催化剂难以与之媲美的高效性和专一性以及催化反应类型的多样性,越来越多的酶已经应用到环境、药物、工业等领域,可以预计未来会有更多的仿生催化剂和工程酶应用到工业生产中。对酶的活性以及特异性机制的充分认识不仅是了解生命活动规律的基础,也是对酶进行修饰改造的前提。但由于酶结构和反应机理的复杂性,仅靠实验方法难以对酶催化反应机理进行全面系统的了解。通过理论与计算化学方法可以从原子水平上揭示酶催化反应的机理,对实验上难以观测到的过渡态及中间体结构进行研究。目前,酶催化反应的理论计算研究是酶催化领域的研究热点之一。理论计算化学方法有很多,研究中需要依据要解决的问题选择合适的方法。本论文主要利用量子力学和分子力学相结合的QM/MM方法对几类金属酶和糖苷酶的催化反应机理进行了较为系统的研究,确定了酶催化反应的最可能路径,得到了反应中间体及过渡态的结构和能量学信息,明确了酶活性中心残基在催化过程中所起的作用,计算结果揭示了这些酶催化反应的机制,对相关实验现象做出解释,并能与现有的一些实验结果很好地吻合,同时也对实验结果做了进一步的说明和补充,对相关酶反应机理的理解解及酶应用奠定了必要的理论基础。主要研究工作如下:(1)AsqJ加双氧酶催化喹啉生物碱合成机理的理论研究喹诺酮结构是多种生物活性分子的骨架结构,4'-甲氧基绿霉素中的4-芳基喹啉片段存在于多种喹啉、喹诺酮生物碱中。近期研究表明曲霉属真菌中的AsqJ酶在4'-甲氧基绿霉素的生物合成中起重要作用,并且AsqJ酶属于非血红素FeⅡ/酮戊二酸依赖加双氧酶。2016年有人报道了 AsqJ与底物结合的晶体结构,并通过实验证实AsqJ酶通过催化两个非耦合的去饱和化与环氧化反应将底物转化为4'-甲氧基绿霉素。基于得到的晶体结构,本文采用QM/MM方法对AsqJ催化的去饱和化反应与环氧化反应进行了研究。计算结果表明,FeⅣ-O复合物需通过一个异构化过程来引发整个催化反应,在去饱和化过程中,FeⅣ=O复合物夺取第一个氢的反应是通过σ-通道进行的,且该反应是催化反应的决速步骤,对应的能垒为19.3 kcal/mol。在环氧化过程中,再次生成的FeⅣ=O复合物对去饱和化中间体的C=C进行加氧反应,对应的能垒为18.1kcal/mol。此外,基于底物N4位缺少甲基会导致催化反应不能进行的实验事实,本文还考察了 N4-甲基对反应的影响,计算发现,N4-甲基的缺失并未直接影响FeⅣ=O的夺氢反应,可能是通过影响酮戊二酸的氧化反应而对催化反应产生影响。这些结果对进一步理解非血红素加双氧酶的反应机理以及喹啉生物碱的生物合成路径提供了坚实的理论基础。(2)咪唑啉酮酸酶催化机理的理论研究组氨酸在生物体内的降解受到严格控制,自然界中组氨酸的降解路径从原核生物到真核生物都是高度保守的。咪唑啉酮酸酶(HutI,EC3.5.2.7)是生物体中组氨酸降解路径中的第三种酶,该酶催化4-咪唑啉酮-5-丙酸(IPA)水解生成L-谷氨酸。前人依据实验结果建议了大致的反应机理,但关于该酶的底物选择性、水分子活化机制等问题依然没有得到解决。本文使用QM/MM方法对IPA的(S)-和(R)-对映体的同分异构体SIPA-1,SIPA-2,RIPA-1和RIPA-2的催化反应进行了研究。计算结果表明,起水解作用的水分子(与锌配位的水分子)是E252残基通过桥连水分子来活化的,且发生在与底物与酶结合之前。在底物结合之后,活化通道就会被底物阻断;另外两个残基(D324,H272)不能活化水分子。HutI只能将IPA的两个(S)-对映体中的SIPA-1转换成L-谷氨酸,其能量势垒为16.6 kcal mol-1。而SIPA-2的转换能垒则为21.9 kcal mol-1。然而,对于两个(R)-对映体来说,RIPA-1能垒更高一些(21.8kcalmol-1),RIPA-2在活性位点的结合作用比sIPA-2更弱。基于计算结果,SIPA-1是HutI最佳的底物,而sIPA-2的水解断键可能需要先转化为SIPA-1才能发生。通过计算我们给出了 HutI催化反应的细节,明确了 HutI酶的底物选择性,为深入了解L-组氨酸在哺乳动物和细菌中的生物降解路径提供了理论基础。(3)氮-乙酰葡糖胺糖苷水解酶催化机理的理论研究糖类在生物体内有许多重要的作用,它除了可以作为结构组分以及提供生物体必要的能量外,还通过对肽聚糖的修饰在免疫响应中扮演重要的角色。肽聚糖(PG)的代谢路径对于细菌的生长至关重要。β-氮-乙酰葡糖胺糖苷酶(NagZ)是肽聚糖代谢路径中一种重要蛋白酶。依据氨基酸顺序以及二级结构的分类,NagZ酶属于糖苷水解酶第三家族(GH3)。然而,最近的实验研究发现NagZ酶是糖苷磷酸化酶而不是糖苷水解酶。为进一步从原子水平上探究NagZ酶的催化机理,本文用QM/MM方法对来源于枯草芽孢杆菌的NagZ酶(BsNagZ)的催化反应进行了研究。计算结果表明,整个催化循环的决速步是糖基化反应,该结论与实验研究相符。糖基化反应对应的能垒为19.3 kcal/mol,该能垒与实验上通过使用类似底物进行反应动力学实验预测的自由能能垒(16.4 kcal/mol)近似。对于去糖基化过程,对水解机理和磷酸化机理的对比研究发现,磷酸化对应的能垒为(1.8kcal/mol),较水解路径的能垒低(17.7kcal/mol),这一结果支持了之前关于NagZ酶为磷酸化糖苷酶的结论。研究还发现,不论是在糖基化还是去糖基化过程中都有类似于羰基正离子过渡态的参与,活性中心底物的构型变化对反应有较大影响,以上理论计算与实验预测吻合。这些计算结果有助于深入理解NagZ酶的催化机理,有助于针对GH3家族β-1,4葡糖胺苷酶的抑制剂的研发。(4)α-1,4-糖苷解酶催化机理的理论研究α-1,4-糖苷裂解酶(GLases,EC 4.2.2.13)是糖苷水解酶家族中独特的一员,它可以特异性的催化糖原、淀粉以及低聚麦芽糖中α-1,4-糖苷键的断裂,从多糖的非还原端断开糖苷键生成1,5-脱水-D-果糖。之前的研究表明,GLases属于构型保持型糖苷裂解酶,在催化反应中涉及到糖苷酶中间体的形成,整个催化循环包括羰基化和去糖基化/消除两个过程。基于最新发表的晶体结构(2X21),本文用QM/MM方法对GLases进行了研究。结果表明,整个催化循环包含五步基元反应。首先天冬氨酸D665做为路易斯酸,向糖苷氧提供一个质子,同时糖苷键发生断裂。然后去质子化的D553残基进攻端基碳形成糖苷酶中间体,该中间体的存在在实验上已得到了论证。在此糖基化过程中普通的保持型糖苷酶是通过协同机理进行的,而该酶的糖基化过程是分步进行的。对于去糖基化/消除反应,我们考虑了反应是在麦芽三糖离开活性位点前发生的或在麦芽三糖离开活性位点之后发生的两种情况。计算结果表明,麦芽三糖的离去有利于去糖基化/消除反应的进行,在这步反应中,去质子化的D553残基可以作为催化碱来夺取糖环上C2位置的质子,并且去糖基化/消除反应中的夺质子反应是整个催化反应的决速步,两种情况对应的势垒分别是20.59和18.53 kcal/mol。糖苷化过程通过D665残基对糖苷氧的质子化来引发反应,糖苷裂解酶的糖基化过程是通过分步反应机理发生的,并且去糖基化/消除反应是整个催化循环的决速步,这些研究结果对于理解α-1,4糖苷酶的催化机理提供了理论依据,并对该类酶的工业应用提供了理论基础。本论文特色和创新之处弄清酶催化反应的机理不仅有助于理解这些蛋白酶的生物学功能,也是对这一高效生物催化剂进行工业应用的重要前提。本论文采用理论化学方法对两类金属酶(AsqJ加双氧酶、咪唑啉酮酸酶)以及两类糖苷酶(BsNagZ酶、α-1,4糖苷裂解酶)的催化反应进行了系统深入的研究,取得了以下重要成果:(1)系统地阐明了 AsqJ加双氧酶催化合成喹啉生物碱的机制,确定了在AsqJ的催化反应中FeⅣ-O的异构化是催化反应的必需步骤,FeⅣ-O复合物的夺氢反应遵循σ-通道机理,分步进行的环氧化反应和去饱和化过程都经历碳自由基中间体,对AsqJ不能催化N4-位不含有甲基的底物类似物的本质原因进行了探讨。(2)预测了咪唑啉酮酸酶(HutI)对四种4-咪唑啉酮-5-丙酸(IPA)的同分异构体的催化活性,阐明了 HutI对底物的选择性本质,解决了实验上存在争议的水分子水分子的活化机制问题,明确了反应中锌离子的作用。(3)从理论阐明了 β-氮-乙酰葡糖胺糖苷酶(NagZ)酶属于磷酸化酶而不是水解酶,即去糖基化过程遵循磷酸化机理,不论是糖基化还是去糖基化过程中都经过类似于羰基正离子过渡态,明确了对该酶底物扭曲其关键作用的残基,验证了糖基化过程是整个催化循环的决速步骤。(4)阐明了 α-1,4糖苷裂解酶的催化机理,给出了催化反应的的最可能路径,明确了糖基化过程是通过分步反应机理发生的,去糖基化/消除反应是整个催化循环的决速步骤,并且验证了实验上预测的反应中涉及到碳正离子中间体以及实验上提出的反应中的亲核试剂做为去糖基化过程中催化碱的机理。
[Abstract]:Enzymes are the basis of all life activities, and the metabolic reactions within almost all cells are inseparable from the involvement of enzymes. It is due to enzyme catalysis that the substances and energy produced in biological metabolism can meet the needs of the organism. The enzyme has the incomparable efficiency and specificity as well as the type of catalytic reaction because of the enzyme's traditional catalyst. More and more enzymes have been applied to the fields of environment, medicine and industry, and more biomimetic catalysts and engineering enzymes can be expected to be applied to industrial production in the future. The full understanding of the activity and specific mechanism of the enzymes is not only the basis for understanding the law of life activities, but also the prerequisite for the modification and modification of the enzyme. Because of the complexity of the enzyme structure and reaction mechanism, it is difficult to fully and systematically understand the mechanism of enzymatic reaction only by experimental methods. Through theoretical and computational methods, the mechanism of enzymatic reaction can be revealed from the atomic level, the structure of the transition state and intermediate structure, which is difficult to be observed in the experiment, is studied. At present, the enzyme catalyzed reaction is used. Theoretical calculation research is one of the hotspots in the field of enzyme catalysis. There are many theoretical computational methods. In this paper, we need to choose the appropriate method according to the problems to be solved. This paper mainly uses the QM/MM method combining quantum mechanics and molecular mechanics to make a more systematic mechanism for the catalytic reaction of several kinds of metal enzymes and glucosidase. The most likely path of enzyme catalysis is determined. The structure and energy information of the reaction intermediate and transition state are obtained. The role of the enzyme active center residues in the catalytic process is clarified. The results of the calculation reveal the mechanism of these enzyme catalytic reactions, explain the related testing phenomena, and can be used with some of the existing experiments. The results are in good agreement, and the experimental results are further explained and supplemented, and the necessary theoretical basis for the understanding of the mechanism of the enzyme reaction and the application of the enzyme are laid. The main research work is as follows: (1) the theoretical study of the synthesis mechanism of quinoline alkaloids by AsqJ and dioxygenase Frame structure, 4- aryl quinoline fragments in 4'- methoxy green mycin exist in a variety of quinoline and quinolone alkaloids. Recent studies have shown that the AsqJ enzyme in Aspergillus fungi plays an important role in the biosynthesis of 4'- methoxy green mycin, and the AsqJ enzyme is a non heme Fe II / ketopamyl acid dependent plus dioxygen enzyme.2016 reported in Asq. The crystal structure of J combined with the substrate has been proved by experiments to prove that the AsqJ enzyme is converted to 4'- methoxy green myxomycin by catalyzing two uncoupled desaturation and epoxidation. Based on the obtained crystal structure, the QM/MM method is used to study the desaturation and epoxidation of AsqJ catalyzed by AsqJ. The calculation results show that F The e IV -O complex needs to initiate the whole catalytic reaction through a isomerization process. During the desaturation, the reaction of the Fe IV =O complex to capture the first hydrogen is carried out through the sigma channel, and the reaction is a quick step of the catalytic reaction. The corresponding energy barrier is 19.3 kcal/mol. in the process of epoxidation, and the Fe IV =O complex is regenerated again. The oxygen reaction to the C=C of the desaturation intermediate, the corresponding energy barrier is 18.1kcal/mol., and the absence of methyl on the substrate N4 will lead to the experimental fact that the catalytic reaction can not be carried out. The effect of the N4- methyl on the reaction is also investigated. It is found that the deletion of N4- methylate does not directly affect the hydrogen capture reaction of the Fe IV =O, which may be passed through Influence of the oxidation of ketopentandiacid on the catalytic reaction. These results provide a solid theoretical basis for further understanding of the reaction mechanism of non heme and dioxygenase and the biosynthesis path of quinoline alkaloids. (2) the theoretical research of the mechanism of imidazolinase catalyzed by histidine is strict in the degradation of the organism. Control, the degradation pathway of histidine in nature is highly conserved from prokaryotes to eukaryotes. HutI (EC3.5.2.7) is the third enzyme in the histidine degradation pathway in organism, which catalyzes the hydrolysis of 4- imidazolinone -5- propionic acid (IPA) to produce L- glutaric acid. However, the substrate selectivity of the enzyme and the activation mechanism of water molecules are still not solved. In this paper, the catalytic reaction of IPA (S) - and (R) - enantiomers SIPA-1, SIPA-2, RIPA-1 and RIPA-2 in enantiomers was studied by QM/MM method. The 252 residue is activated by a bridge with water molecules and occurs before the substrate is combined with the enzyme. After the substrate binding, the activation channel is blocked by the substrate; the other two residues (D324, H272) can not activate the water molecule.HutI only to convert the SIPA-1 in the enantiomer to the L- Glutamic acid, and the energy barrier of the IPA is 16.6 kcal mol-1. and S. The conversion energy barrier of IPA-2 is 21.9 kcal mol-1., however, for two (R) enantiomers, RIPA-1 can be higher (21.8kcalmol-1), RIPA-2 is weaker in the binding of the active site than sIPA-2. Based on the calculation, SIPA-1 is the best substrate for HutI, and sIPA-2 water disconnection may need to be converted to SIPA-1 to occur first. The details of the HutI catalytic reaction are given and the substrate selectivity of the HutI enzyme is clarified. The theoretical basis for understanding the biodegradation path of L- histidine in mammals and bacteria is provided. (3) the theoretical study of the catalytic mechanism of N-acetylglucosamine hydrolase The metabolic pathway of peptidoglycan (PG) is important for the growth of the bacteria. Beta n-acetylglucosinase (NagZ) is an important protease in the pathway of peptidoglycan metabolism. The NagZ enzyme belongs to the third family of glucoside hydrolase (GH3). However, the recent experimental study found that the NagZ enzyme is glucoside phosphorylase instead of glucoside hydrolase. In order to further explore the catalytic mechanism of NagZ enzyme from the atomic level, the QM/MM method has been used in QM/MM to promote the NagZ enzyme (BsNagZ) derived from Bacillus subtilis (BsNagZ). The results show that the quick step of the whole catalytic cycle is a glycosylation reaction, which is consistent with the experimental study. The corresponding energy barrier of the glycosylation reaction is 19.3 kcal/mol, and the energy barrier is similar to the free energy barrier (16.4 kcal/mol) predicted by the experimental kinetic experiment by using similar substrates. In the process of glycosylation, a comparative study of the mechanism of hydrolysis and phosphorylation found that the corresponding energy barrier of phosphorylation was (1.8kcal/mol), which was lower than the energy barrier of the hydrolytic pathway (17.7kcal/mol). This result supported the previous conclusion that the NagZ enzyme was phosphorylated glucosidase. With the participation of the transition state of carbonyl positive ions, the configuration changes of the active center substrates have great influence on the reaction, and the theoretical calculation coincides with the experimental prediction. These results are helpful to understand the catalytic mechanism of NagZ enzyme and help the development of the inhibitor of the GH3 family beta Glucosaminidase. (4) the alpha -1,4- glycosidase catalytic machine The theoretical study of the theoretical study of alpha -1,4- glycoside lyase (GLases, EC 4.2.2.13) is a unique member of the glycoside hydrolase family. It can specifically catalyze the fracture of glycosides, starch and the alpha -1,4- glycosides of oligosaccharides, and disconnect the glycosides from the non reductive ends of the polysaccharides to produce 1,5- dehydrated -D- fructose. Previous studies have shown that GLases belongs to the configuration. The retention type glucoside lyase is involved in the formation of glucosidase intermediates in the catalytic reaction. The whole catalytic cycle includes carbonylation and glycosylation / elimination of two processes. Based on the latest published crystal structure (2X21), the QM/MM method is used to study GLases. The results show that the whole catalytic cycle contains five step reaction. Aspartate D665 is a Lewis acid, which provides a proton to glycoside oxygen, while the glycosidic bond breaks. Then the protonated D553 residues attack the end group carbon to form a glycosidase intermediate. The existence of the intermediate has been demonstrated experimentally. In this glycosylation process, the common preserved glucosidase is carried out through synergistic mechanism. The glycosylation process is carried out step by step. For the deglycosylation / elimination reaction, we consider that the reaction occurs before the malt three sugar leaves the active site or after the malt three sugar leaves the active site. The results show that the departure of the malt three sugar is beneficial to the deglycosylation / elimination of the reaction, in this case, the deglycation of malt three sugar is beneficial to the deglycosylation / elimination reaction. In the step reaction, the deprotonated D553 residue can be used as a catalytic base to capture protons on the C2 position on the sugar ring, and the protonic reaction in the deglycosylation / elimination reaction is the quick step of the whole catalytic reaction. The corresponding potential barriers in the two cases are 20.59 and 18.53 kcal/mol. Glucosidation, respectively, by the protonation of glucoside oxygen through the D665 residue. The glycosylation process of glucoside lyase occurs by step reaction mechanism, and glycosylation / elimination reaction is a quick step for the whole catalytic cycle. These results provide a theoretical basis for understanding the catalytic mechanism of alpha -1,4 glucosidase, and provide a theoretical basis for the industrial application of this kind of enzyme. Making clear the mechanism of enzymatic reaction is not only helpful to understand the biological function of these proteases, but also an important prerequisite for the industrial application of this highly efficient biocatalyst. This paper uses the theoretical chemistry method for two kinds of metal enzymes (AsqJ plus dioxygenase, imidazolidase) and the two kind of glucosidase (BsNagZ enzyme, alpha -1,4 sugar). The catalytic reaction of glycoside lyase has been systematically studied, and the following important achievements have been obtained: (1) the mechanism of AsqJ and dioxygenase catalyzed synthesis of quinoline alkaloids is systematically clarified. The isomerization of Fe IV -O in the catalytic reaction of AsqJ is a necessary step for catalytic reaction, and the hydrogen capture reaction of Fe IV -O complex follows the sigma channel mechanism. The cyclic oxidation reaction and desaturation process all undergo carbon free radical intermediates, and the essential reason for AsqJ's inability to catalyze the substrate analogues without methyl N4- is discussed. (2) the catalytic activity of imidazolininase (HutI) on the isomers of the four 4- imidazolinone -5- propionic acid (IPA) is predicted, and the HutI pair is clarified. The selective nature of the substance has solved the activation mechanism of water molecules that have been disputed experimentally and clarified the effect of zinc ions in the reaction. (3) it is explained from theory that beta nitrogen acetyl glucosidase (NagZ) enzyme belongs to phosphorylase rather than hydrolase, that is, the process of glycosylation follows phosphorylation mechanism, whether glycosylation or In the process of deglycosylation, the transition state similar to carbonyl positive ion is passed, and the key effect on the enzyme substrate is identified. The glycosylation process is the quick step of the whole catalytic cycle. (4) the catalytic mechanism of the alpha -1,4 glycoside lyase is clarified, the most possible path of the catalytic reaction is given, and the glycosylation process is clearly defined. It is by step reaction mechanism that de glycosylation / elimination reaction is the quick step of the whole catalytic cycle, and it is verified that the experimental reaction involves the carbon cation intermediate and the nucleophilic reagents in the experimental reaction as the mechanism of the catalytic alkali in the process of glycosylation.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O629.8;O643.31

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