Theoretical Study On The Catalytic Mechanisms Of Typical Environment Related Enzyme Systems

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.