【作者】 李延伟；

【导师】 张庆竹；

【作者基本信息】 山东大学 ， 环境科学， 2014， 博士

【摘要】 随着生物化学技术的快速发展,酶作为一种高效、专一且反应条件温和的生物催化剂,被越来越多的科研工作者用于研究、治理我国日益严峻的环境污染问题。酶与环境的关系十分密切,人们可以利用生物酶降解环境中的污染物,或者通过酶催化以及仿生催化工艺减少能源消耗和污染物排放,以达到清洁化生产的目的。此外,研究环境污染物对生物体内酶的抑制机理以及生物毒理学效应,可以为制定环境标准和开展环境保护工作提供科学依据。目前实验上研究酶催化反应机理的手段多种多样,包括红外光谱法、分子散射法、X射线衍射法、核磁共振法、酶促反应动力学法、同位素标记法等,然而这些手段无法详细的描述酶的催化反应过程,限制了酶学研究的发展。近年来兴起的量子力学与分子力学联用(QM/MM)方法能够很好的描述酶的催化反应过程,对酶催化领域产生了重大影响,已成为不可或缺的强有力的研究手段之一。本论文综合使用分子动力学(MD)方法以及QM/MM方法研究了几种与环境问题密切相关的酶体系的催化反应机理,揭示了酶催化反应过程的详细信息,验证、解释并补充了相关实验结果,促进了酶分子催化体系方法的发展及其在环境领域的应用。一、乙酰胆碱酯酶体系人体以及很多动物体内都存在乙酰胆碱酯酶,其主要作用是降解神经递质乙酰胆碱以终止突触传递。而有些有机磷类化合物(如化学武器制剂塔崩、杀虫剂甲胺磷)能通过抑制、老化等作用使乙酰胆碱酯酶失活,从而造成乙酰胆碱在人体内过度堆积,继而引发肌肉麻痹与呼吸困难,最后导致死亡。目前对于有机磷化合物对乙酰胆碱酯酶的抑制机理已经研究的较为深入,但是对于有机磷化合物抑制的乙酰胆碱酯酶的老化机理、药物活化机理以及自发活化机理研究的较少。由于塔崩抑制的乙酰胆碱酯酶不能自发活化,因而本论文以塔崩为例,使用MD方法以及QM/MM方法研究了有机磷化合物抑制的乙酰胆碱酯酶的老化机理及肟类化合物的药物活化机理,发现塔崩的老化过程是通过C-O键断裂而不是通过P-N键断裂完成的,并且通过研究CH2NO、HLO-7两种肟类解毒剂对塔崩抑制的乙酰胆碱酯酶的活化机理,建立了判定药物分子是否有药效的两条基本判据,对新型药物的设计有指导意义。由于杀虫剂甲胺磷抑制的乙酰胆碱酶的自发活化速率远高于其老化速率,因而,它是一个很好的研究有机磷类化合物抑制的乙酰胆碱酶自发活化反应机理的模型。QM/MM计算结果显示自发活化反应过程中存在一种三角双锥性质的中间体,并发现甲胺磷的不同光学异构体的自发活化能力不同。二、黄素腺嘌呤二核甘酸依赖性亚硝基合酶体系由碳样小单孢菌(Micromonospora carbonacea)中的基因oof36编译的黄素腺嘌呤二核甘酸依赖性亚硝基合酶(ORF36)能够催化复杂化合物中亚硝基基团的合成,在制药、食品及精细化工等工业领域有巨大的应用价值。本论文以药物前体物胸苷二磷酸-L-型万古胺为例,使用QM/MM方法深入研究了酶ORF36催化生成亚硝基基团的反应机理,以期为后续的仿生催化、清洁生产提供理论指导。计算结果显示：(1)第二次氧化反应分为三个基元反应步骤且第一步羟基转移反应为速控步。(2)产物亚硝基基团中的氧原子来源于第二次氧化而不是第一次氧化过程中的氧分子。(3)通过研究活性中心附近十八种氨基酸对羟基转移反应的静电影响发现,Ser162对速控步反应的抑制作用最为明显,而氨基酸Leu134和Gln376对速控步反应的促进能力最明显。三、联苯间位产物水解酶体系间位产物水解酶属于α/β-水解酶家族,能够催化芳香族化合物的C-C键水解,对全球碳循环以及人体健康有着重要影响。其中,间位产物水解酶BphD在联苯、多氯联苯(环境中的残留量大于28万吨)等环境污染物的降解过程中的地位非常重要。本论文以联苯为模型化合物,使用QM/MM方法探索了酶BphD催化降解联苯、多氯联苯类污染物的反应机理。研究结果发现：(1)Ser112-Glu237-His265催化三元体参与到了野生型酶BphD的催化酰化反应过程。(2)只有Ser112参与到了酶H265A变体的催化酰化过程,因而野生型酶与H265A变体的催化反应机理不同。(3)在酰化反应完成之后,产物HPD在BphD活性中心占有的空间由三个水分子填补,且其中一个水分子直接参与到接下来的脱酰化反应过程。(4)通过分析活性中心部位氨基酸对速控步反应的静电影响,验证并解释了突变Phe175、Arg190、Trp266将降低催化反应速率这一实验结果。(5)本论文同样发现了G1y42、Met113、Leu156、Phe239对速控步反应速率的影响,并认为针对Phe239进行饱和突变或许能发现提高酶BphD催化降解速率的变体酶。四、谷胱甘肽转移酶体系全世界每年约有140万人因感染疟疾这一蚊虫传播疾病而死亡,人们通常通过喷洒DDT的办法来消灭蚊虫以达到控制疟疾传播的目的。可是近年来发现某些蚊虫体内的谷胱甘肽转移酶能够将高毒性的DDT转化为低毒性的DDE,从而产生了DDT抗性。因而,为了保护人类健康,亟需研发一种能够替代DDT的杀虫剂。本论文首先使用QM/MM方法分别研究了谷胱甘肽转移酶agGSTe2催化DDT降解为DDE的两种可能的机理：质子转移机理和GS-DDT共聚物机理。结合实验数据,计算结果确定了agGSTe2解毒DDT的机理为质子转移机理。构建了一系列DDT类似物,并发现其中一个类似物,(BrC6H4)2CHCC13,抵抗agGSTe2催化解毒反应的能力最佳。本论文的研究结果为设计新型杀虫剂以控制疟疾传播提供了理论模型以及理论指导。五、氟乙酸盐脱卤素酶体系含氟化合物的大量生产和使用给环境带来了巨大的压力,然而目前发现的能够降解含氟化合物的酶十分稀少。研究发现氟乙酸盐脱卤素酶能够催化降解氟乙酸类化合物(该化合物在国内外被广泛用于消灭鼠害)。本论文使用QM/MM方法从分子水平上研究了氟乙酸盐脱卤素酶催化C-F键断裂的催化反应机理,探索了活性中心部位His155不同结构(Hsd或Hse)对酶催化反应的影响,发现氟乙酸盐脱卤素酶分子更倾向于通过Hse结构催化C-F键的断裂。该研究成果将有助于建立其它含氟化合物降解的理论模型。更多还原

【Abstract】 Due to the rapid development of biochemical technology, enzymes which enjoy high efficiency, specificity, and mild reaction condition properties are more and more widely used in investigating and managing the increasingly serious environmental pollution of China. Enzymes are closely related to the environmental issues. They can be either applied in degrading the environmental pollutants or used in biomimetic catalysis for clean production purpose. In addition, studying the inhibition mechanism and toxicological effects of environmental pollutants toward the functional enzymes can provide scientific basis for environmental protection. Many experimental methods have been established to investigate the enzyme catalysis, such as infrared spectroscopy, molecular scattering method, nuclear magnetic resonance method, enzyme kinetics, and isotope labelling method. However, these methods cannot describe the detailed catalytic itinerary of enzymes which limits the development of enzymology. The quantum mechanics/molecular mechanics (QM/MM) approach is by now established as a valuable and irreplaceable tool in modelling the catalytic system of enzymes.The molecular dynamics (MD) and QM/MM approaches are used in this dissertation to study the catalytic mechanism of several environmental related enzymatic systems. The results revealed the catalytic itinerary of the studied enzymes. The calculated results also verified, explained, or supplemented the experimental ones. Thus, this dissertation may promote the use of QM/MM method in enzymatic systems and the application of QM/MM method in the environmental related field.1. Catalytic system of acetylcholinesteraseAcetylcholinesterase (AChE) in human or animals hydrolyzes the neurotransmitter acetylcholine (ACh) to terminate synaptic transmission. However, many organophosphorus compounds can hinder its hydrolysis function towards Ach, such as chemical warfare agent tabun and insecticide methamidophos. Successive accumulation of Ach will paralyze neurotransmission and eventually lead to muscle fasciculation and respiratory failure. Without injecting proper antidote immediately, people may die soon after the poisoning. So far, the inhibition mechanism of organophosphorus compounds toward AChE has been extensively studied. However, the studies on the aging mechanism, antidote induced reactivation mechanism and spontaneous reactivation mechanism of organophosphorus compounds toward AChE are limited.Since tabun inhibited AChE cannot be spontaneously reactivated, we only focused on studying its aging mechanism and antidote induced reactivation mechanism. The calculated results revealed that the aging mechanism is accomplished through the C-O bond scission rather than P-N bond scission. In addition, we established two criteria for forecasting the reactivate efficacy of newly designed antidote on the basis of the CH2NO-and HLO-7induced reactivation processes.The system of methamidophos inhibited AChE serves as a perfect model for studying the spontaneous reactivation mechanism of organophosphorus compounds inhibited AChE. The QM/MM results show that there is an intermediate with a trigonal bipyramid characteristic. In addition, different optical isomers of methamidophos show different spontaneous reactivation abilities.2. Catalytic system of a FAD-dependent nitrososynthaseA FAD-dependent nitrososynthase (ORF36) encoded by gene orf36from Micromonospora carbonacea mediates the synthesis of nitroso group of macromolecular compounds, and shows great potential in the application of pharmaceutical, finechemical and food industries. In this dissertation, we used QM/MM approaches to investigate the catalytic mechanism of ORF36toward a drug candidate (TDP)-L-epi-vancosamine. The results serve as excellent model for the future biomimetic synthesis of (TDP)-L-epi-vancosamine or other nitroso group containing macromolecular compounds. The QM/MM results reveal that (1) The catalytic mechanism of the second oxidation step of ORF36consists of three elementary steps and the first step is rate-determining.(2) Oxygen atom which comes from the second oxidation step is found in the product.(3) By studying the electrostatic influence of18amino acids, Ser162was found to suppress the hydroxylation reaction most, while Leu134and Gln376was found to contribute equally in facilitating it.3. Catalytic system of meta-cleavage product hydrolasesThe meta-cleavage product (MCP) hydrolases are members of α/β-hydrolase superfamily, which catalyze C-C bond hydrolysis of aromatic compounds and are implicated in processes like global carbon cycle and human health. For instance, a MCP hydrolase BphD from biphenyl degradation pathway is crucial in the mineralization of polychlorinated biphenyls (PCBs). There is about2.8×108kg PCBs in mobile environment reservoirs, causing a severe environmental concern. In this dissertation, QM/MM calculations on investigating the catalytic mechanism of BphD toward biphenyl (model compound) were carried out. The following results are obtained:(1)Ser112-His265-Asp237is involved in wild BphD acylation process.(2) Only Ser112is involved in H265A mutant acylation process.(3) Three water molecules are found in the active pocket after HPD release, and one of which is involved in the deacylation process.(4) Previously reported roles of residue Phe175, Arg190, and Trp266in the rate-determining step of wild BphD are further elucidated by our computational results.(5) The electrostatic analysis reveal the significant roles of some unnoticed residues, such as Gly42, Metll3, Leu156, and Phe239, the results thus highlight new promising experimental targets in improving the catalytic efficiency of wild BphD.4. Catalytic system of glutathione transferasesMalaria is a most severe insect transmitted disease with at least1.4million deaths per year. Organochlorine insecticide DDT plays a prominent role in controlling the population of malaria vector mosquitoes since1950s. However, resistance to DDT has developed in some mosquito species (e.g. Anopheles Gambiae), raising the threat of malaria to humans. A mechanistic understanding of the detoxifying of GSTs towards DDT is critically warranted to understand the impact of DDT resistance and to develop more effective novel insecticides. We investigated the detoxification reaction of agGSTe2towards DDT by using QM/MM method. The results reveal that the proton transfer mechanism is more feasible than the GS-DDT conjugation mechanism. On the basis of the structure of DDT, structure2,(BrC6H4)CHCCl3, is the best candidate among all the tested structures in resisting the detoxification of enzyme agGSTe2. Our work serves as a theoretical model for designing new insecticides in controlling insect transmitted disease malaria.5. Catalytic system of fluoroacetate dehalogenaseThe production and use of fluorochemicals brought great pressure to the environment, however, few enzymes are found to be able to degrade those fluorochemicals. An enzyme named fluoroacetate dehalogenase (FAcD) is capable of degrading fluoroacetate (FAc), which is wildly used as rodenticide in many countries. We investigated the catalytic mechanism of FAcD toward FAc by using QM/MM method to establish a pioneer model for studying the degradation mechanism of other fluorochemicals. The results also indicate that structural differences of His155(Hsd or Hse) influence the reaction barrier of C-F bond cleavage process and FAcD prefers Hse155when catalyzing FAc.更多还原