本文選題：密度泛函理論 + 催化機(jī)理��； 參考：《哈爾濱工業(yè)大學(xué)》2017年碩士論文

【摘要】：有機(jī)磷化合物作為一種廣泛應(yīng)用的高效殺蟲劑以及一種化學(xué)武器,其對(duì)人體的傷害是巨大的。目前,在解毒有機(jī)磷中毒方面使用較多的化學(xué)試劑為環(huán)己酮肟,但其效率較低。近年來,隨著酶學(xué)在生物催化中的發(fā)展,設(shè)計(jì)一種能高效解毒有機(jī)磷中毒的酶分子來代替化學(xué)試劑的使用越來越受到人們的關(guān)注。DFPase來源于槍烏賊的中樞神經(jīng)系統(tǒng),而PON1則由哺乳類動(dòng)物的肝臟合成,兩者都能夠有效的水解有機(jī)磷化合物并使P-F鍵、P-O鍵以及P-CN鍵。DFPase與PON1兩種酶功能上相似,結(jié)構(gòu)上具有細(xì)微差別,二者具有一定的進(jìn)化相關(guān)性。研究二者催化機(jī)理間的進(jìn)化關(guān)系能夠?yàn)榈鞍踪|(zhì)工程研究高性能解毒劑提供一定的理論依據(jù)。在本文的研究中,采用雜化密度泛函理論分別研究了DFPase和PON1的催化機(jī)理。對(duì)DFPase的研究中,分別設(shè)計(jì)了Asp229和H2O作為親核試劑進(jìn)攻底物磷中心的反應(yīng)路徑。計(jì)算結(jié)果顯示,不論是與Ca2+配位的Asp229還是由其激活的H2O作為親核試劑,都能形成對(duì)底物的親核進(jìn)攻,其反應(yīng)的自由能能壘分別是14.8 kcal/mol和6.0 kcal/mol,相比之下,由H2O作為親核試劑進(jìn)攻底物磷中心是最有利的反應(yīng)途徑,而Asp229不論是作為親核試劑還是廣義堿都對(duì)水解反應(yīng)起著非常重要的作用。此外,對(duì)于所形成的磷酸酶中間體,Ca2+六配位結(jié)構(gòu)是最優(yōu)構(gòu)象。而在整個(gè)水解反應(yīng)中,Asn120和Asn175則通過與離去基團(tuán)形成氫鍵而促進(jìn)其離去�；趯�(duì)DFPase催化機(jī)理的研究,本文設(shè)計(jì)了兩種由H2O作為親核試劑的PON1模型,分別是野生型PON1和突變型PON1模型(在野生型PON1模型基礎(chǔ)上將Asn270突變成H2O以模擬DFPase的結(jié)構(gòu)),之后分別將底物DFP和Paraoxon與兩種模型復(fù)合從而研究PON1與DFPase催化機(jī)理的進(jìn)化關(guān)系。研究結(jié)果表明,將Asn270突變成H2O后,提高了PON1對(duì)底物DFP的活性,相應(yīng)的降低了對(duì)Paraoxon底物的活性;此外,這種突變也提高了PON1對(duì)DFP催化產(chǎn)物的穩(wěn)定性。本論文的研究不僅解釋了DFPase和PON1催化水解有機(jī)磷試劑的機(jī)理,同時(shí),也提出了新的酶催化機(jī)理的進(jìn)化觀點(diǎn),為實(shí)驗(yàn)室合成新的解毒有機(jī)磷試劑的藥物提供了指導(dǎo)意義。
[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.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O643.31;TQ421

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本文編號(hào)：1799942