The Oretical Studies On Mechanisms Of Methyl Parathion Hydrolase Hydrolysis-Catalyzed Reaction

Methyl parathion hydrolase(MPH, EC 3.1.8.1), which is able to effectively catalyze the hydrolysis of the toxic organophosphorus compounds through specifically breaking their phosphoester bonds. MPH prefers to catalyze the substrate methyl parathion(MPS), and can also effectively catalyze methyl paraxon(MPO). In this thesis, we focus on the MPH from the soildwelling bacterium Pseudomonas sp. WBC-3(MPH-WBC3), which is dinuclear zinc enzyme and belongs to the beta-lactamase protein family.Based on the MPH-WBC3 crystal structure, the catalytic mechanism of MPH was investigated by using density functional theory(DFT) method. According to the structural characteristics of substrate and the active site, two possible models were designed. One is the hydroxide(μ-OH–) bridging two Zn2+ in the active site of MPH attacking on the phosphorus atom from para position of the leaving group; another is μ-OH– attacking the phosphorus atom from ortho position. For MPH catalyzing MPS, the calculation results show that it is feasible to take place of the nucleophile μ-OH– attacking on the phosphorus atom from the ortho position. It is a two step reaction. For the first step, Asp151 as a general base abstracted a proton from μ-OH– and let it activated; for the second step, the nucleophile attacked on the phosphorus center and made the P–O bond between the leaving group and phosphorus atom break. At the same time, Asp151 as a general acid provided a proton to the leaving group and made it easier leave. For MPH catalyzing MPO, μ-OH– para position attacking on MPO is more favorable. Asp151 as a general base abstracted a proton from μ-OH– and made it activated to attack on the phosphorus atom; meanwhile the P–O bond broke and the nitrophenol anion left. Our calculation results show that MPH is able to efficiently catalyze both MPS and MPO. Both the reactions are exothermic and quite easy to occur as their energy barriers less than 3.0 kcal/mol. These results agree well with the experimental phenomena.Our theoretical results provide the details of the reaction mechanisms of MPH catalyzed MPS and MPO, and the interaction changes among the residues, metal centers, and substrates during the reaction processes. These will provide clues for the enzyme design and modify.