从PON1到DFPase催化活性机理的进化关系的理论研究

发布时间：2018-04-25 05:21

本文选题：密度泛函理论 + 催化机理　；参考：《哈尔滨工业大学》2017年硕士论文

【摘要】：有机磷化合物作为一种广泛应用的高效杀虫剂以及一种化学武器,其对人体的伤害是巨大的。目前,在解毒有机磷中毒方面使用较多的化学试剂为环己酮肟,但其效率较低。近年来,随着酶学在生物催化中的发展,设计一种能高效解毒有机磷中毒的酶分子来代替化学试剂的使用越来越受到人们的关注。DFPase来源于枪乌贼的中枢神经系统,而PON1则由哺乳类动物的肝脏合成,两者都能够有效的水解有机磷化合物并使P-F键、P-O键以及P-CN键。DFPase与PON1两种酶功能上相似,结构上具有细微差别,二者具有一定的进化相关性。研究二者催化机理间的进化关系能够为蛋白质工程研究高性能解毒剂提供一定的理论依据。在本文的研究中,采用杂化密度泛函理论分别研究了DFPase和PON1的催化机理。对DFPase的研究中,分别设计了Asp229和H2O作为亲核试剂进攻底物磷中心的反应路径。计算结果显示,不论是与Ca2+配位的Asp229还是由其激活的H2O作为亲核试剂,都能形成对底物的亲核进攻,其反应的自由能能垒分别是14.8 kcal/mol和6.0 kcal/mol,相比之下,由H2O作为亲核试剂进攻底物磷中心是最有利的反应途径,而Asp229不论是作为亲核试剂还是广义碱都对水解反应起着非常重要的作用。此外,对于所形成的磷酸酶中间体,Ca2+六配位结构是最优构象。而在整个水解反应中,Asn120和Asn175则通过与离去基团形成氢键而促进其离去。基于对DFPase催化机理的研究,本文设计了两种由H2O作为亲核试剂的PON1模型,分别是野生型PON1和突变型PON1模型(在野生型PON1模型基础上将Asn270突变成H2O以模拟DFPase的结构),之后分别将底物DFP和Paraoxon与两种模型复合从而研究PON1与DFPase催化机理的进化关系。研究结果表明,将Asn270突变成H2O后,提高了PON1对底物DFP的活性,相应的降低了对Paraoxon底物的活性;此外,这种突变也提高了PON1对DFP催化产物的稳定性。本论文的研究不仅解释了DFPase和PON1催化水解有机磷试剂的机理,同时,也提出了新的酶催化机理的进化观点,为实验室合成新的解毒有机磷试剂的药物提供了指导意义。
[Abstract]:Organophosphorus compounds are widely used as a highly effective insecticide and a chemical weapon, their harm to human body is great. At present, cyclohexanone oxime is widely used in detoxification of organophosphorus poisoning, but its efficiency is low. In recent years, with the development of enzymology in biocatalysis, the design of an enzyme molecule which can detoxify organophosphorus poisoning to replace the chemical reagent has attracted more and more attention. DFPase is derived from the central nervous system of squid. However, PON1 was synthesized from mammalian liver. Both of them could effectively hydrolyze organophosphorus compounds and make P-F bond, P-O bond and P-CN bond. DFPase and PON1 had similar function and slight difference in structure. Both of them had a certain evolutionary correlation. The study of the evolutionary relationship between them can provide a theoretical basis for the study of high performance antidote in protein engineering. In this paper, the catalytic mechanisms of DFPase and PON1 were studied by hybrid density functional theory. In the study of DFPase, the reaction paths of Asp229 and H _ 2O as nucleophilic reagents to attack the phosphorous center of substrate were designed, respectively. The results show that either the Asp229 coordinated with Ca2 or the H 2O activated by it can form a nucleophilic attack on the substrate. The free energy barrier of the reaction is 14.8 kcal/mol and 6.0 kcal / mol, respectively. It is the most favorable way to attack the phosphorous center of substrate by H _ 2O as nucleophilic reagent, and Asp229 plays a very important role in hydrolysis both as nucleophilic reagent and generalized base. In addition, the hexacoordination structure of Ca 2 2 is the optimum conformation for phosphatase intermediates. In the whole hydrolysis reaction, Asn 120 and Asn175 promote the separation by forming hydrogen bond with the group leaving. Based on the study of the catalytic mechanism of DFPase, two PON1 models with H2O as nucleophilic reagent were designed. On the basis of wild type PON1 model, Asn270 was mutated into H2O to simulate the structure of DFPase, and then the substrate DFP and Paraoxon were combined with the two models to study the evolutionary relationship of PON1 and DFPase catalytic mechanism. The results showed that the activity of PON1 to substrate DFP was increased and the activity of Paraoxon substrate was decreased by mutating Asn270 to H2O. In addition, this mutation also improved the stability of PON1 to DFP catalytic products. The research in this paper not only explains the mechanism of hydrolysis of organophosphorus reagents catalyzed by DFPase and PON1, but also puts forward a new evolutionary viewpoint of enzymatic catalytic mechanism, which provides guidance for the synthesis of new detoxifying organophosphorus reagents in laboratory.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O643.31;TQ421

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