Theoretical Study Of Organophosphorus Toxicology And Organochlorine Degradation In Enzyme Systems

Enzymes are a kind of biomacromolecules with catalytic activity and specific spatial conformation.They have been researched and applied in many areas such as industrial and agricultural production,medical and health,due to the features of high efficiency,specificity and temperate reaction condition.The environmental application of enzymes is attracting more and more attention nowadays,as the environmental issue becomes more and more serious.There are close relationships between enzyme and environment.The environmental pollutant can cause acute hazard to organisms through damaging the structure and function of enzyme directly.On the other hand,many kinds of enzymes can degrade environmental pollutants,which can remove pollutants and mitigate the environmental crisis.So,investigating the toxicological effect of environmental pollutants,recognizing the poisoning mechanism of enzymes can provide more valuable instructions in drug design and detoxification.Meanwhile,enzymes can be widely used in pollution detection and abatement due to their degrading capability to pollutants.The enzymatic structure,function and reaction mechanism can be investigated by many experimental installations at present,but they cannot describe the detailed reaction process,which limits the development of enzymology.The quantum mechanics/molecular mechanics(QM/MM)method can study the enzyme process in atomic scale and therefore become an important tool in the study of biological macromolecular reaction system.In the present dissertation,organophosphorus toxicology and organochlorine degradation in enzymatic system were investigated with the aid of molecular dynamics(MD)and QM/MM method.The details of reaction process were provided;the enzyme activity and reaction characteristic were discussed;some related experimental results were verified and explained.As a consequence,the present dissertation can enrich the knowledge of enzyme and promote its application in environment.1.Reactivation and aging of phosphorylated acetylcholinesteraseOrganophosphates can phosphorylate acetylcholinesterase causing the dysfunction of acetylcholine decomposition and further destroying the nerve conduction.The subsequent reaction for the phosphorylated acetylcholinesterase can be either reactivation or aging.Studies for the two subsequent reactions are insufficient.Especially for the aging reaction,the water molecule that can initiate the reaction is still undefined and the amino acid that can polarize the water molecule is also uncertain.Taking paraoxon as an example of organophosphate,this dissertation investigated the reactivation and aging process of phosphorylated acetylcholinesterase with the aid of a QM/MM method.Results indicated that a water molecule polarized by His447 was involved in the reactivation process,while the aging process was initiated by another water molecule which was polarized by Glu202.The phosphorous atom in paraoxon was attacked during the reactivation process and the a-carbon atom of alkoxyl in paraoxon was involved in the aging process.Furthermore,Glyl22 was proved to contribute the most in facilitating both of the two processes,while the main obstacle in reactivation process was Gly121 and the major obstruction in aging process was Tyr133.The present dissertation can provide new methods for gaining insight into the structure and function of enzyme.2.Aging of phosphorylated acetylcholinesterase with different protonated state of histidineThe amino acid Glu202 has been proved to involve in the aging of phosphorylated acetylcholinesterase while His447 do not participate in it directly.Even so,its spatial position and electrical character determine that His447 is bound to exert an influence on the aging process.Furthermore,pKa of the protonated imidazole ring in His447 is approximately 6.5 under the general physiological conditions.It implies that the protonated His447 will go through partial ionization under the general physiological conditions and can act as proton donor and acceptor.So,it is interesting to investigate the aging of the phosphorylated acetylcholinesterase with His447 ineither protonated or unprotonated state.By using a QM/MM method,this dissertation employed dimethyl phosphate inhibited acetylcholinesterase to explore the effect of different His447 protonated states on the aging reaction.Results revealed that the unprotonated state was more favorable to the aging of dimethyl phosphorylated acetylcholinesterase.The proton in protonated His447 weakens the nucleophilic ability of Glu202 and further obstructs the nucleophilic water molecule to attack the carbon atom of organophosphorus.So,increasing the concentration of protonated His447 by changing the acid-base balance can delay the aging reaction,which can get more time for organophosphorus detoxification.Also,increasing the concentration of unprotonated His447 can promote the secondary poisoning that enhances the toxicity of organophosphorus pesticide.This dissertation boldly presented a new idea for pesticide or antidote designing and also gave new understanding about structure and function of acetylcholinesterase.3.Dehydrochlorination mechanism of γ-hexachlorocyclohexaneAs a kind of organochlorine insecticide,y-hexachlorocyclohexane is also an important environment pollutant,which can eliminate two hydrogen chloride molecules with the degradation of hexachlorocyclohexane dehydrochlorinase(LinA).Structure and function analysis indicated only the adjacent trans-diaxial H/Cl pair that can be eliminated by LinA.However,the product of the first dehydrochlorination cannot meet the condition of LinA-catalyzed second dehydrochlorination.As a result,the present dissertation modified the LinA-catalyzed biotransformation pathway of y-hexachlorocyclohexane,suggesting that a conformational transition process should be occurred in the middle of the two dehydrochlorination processes.This assumption was confirmed through the theoretical simulation study.Also,this dissertation theoretically verified the existence of the putative short-lived product and described the biological significance of two dehydrochlorination processes by Sphingomonadaceae microorganism.Furthermore,it also revealed that Phe68 facilitates the dehydrochlorination of y-hexachlorocyclohexane,whereas Leu21 and Cys71 suppress it.It can be a valuable base for rational design of mutants of dehydrochlorinase LinA with a more efficient activity towards the degradation of y-hexachlorocyclohexane.4.Enantioselective hydrolysis dechlorination mechanism of 1,2-dichloropropaneHaloalkane dehalogenase LinB has been widely used in bioremediation owing to its broad substrate specificity and simple degrading condition.A weak but detectable activity of LinB was found when degrading 1,2-dichloropropane(DCP)which is an organochlorine pollutant with extremely strong environmental stability.Besides,DCP is a racemic mixture with two optical isomers and whether LinB is enantioselective towards the dechloridation of DCP is still uncertain.This dissertation investigated LinB-catalyzed hydrolytic dechlorination of two DCP enantiomers at atomic scale.It demonstrated that LinB can exhibit high enantioselectivity on the degradation of DCP and biodegradability of(R)-isomer is much higher than(S)-isomer.Further spatial position analysis revealed that the different orientations of the two enantiomers generate their specific microenvironments.As a consequence,another water molecule is allocated to the active site of(S)-DCP-LinB reaction system.It can obstruct the dechlorination of(S)-DCP and therefore is considered to be a crucial factor for the enantioselectivity of LinB towards the degradation of DCP.The electrostatic influence analysis reveals that degradation of the two DCP isomers are all suppressed by residue Gly37.Future mutation studies for improving the degradation efficiency of haloalkane dehalogenase LinB can focus on mutating the amino acids of Gly37.