Theoretical Studies On Catalysis Mechanism Suggesting An Evolutional Trend From Paraoxonase Activity To DFPase Activity

The organic phosphorus compound,as a widely used high-performance pesticide and a chemical weapon,does great damage to human body.The most used chemical reagents for detoxicating the organic phosphorus is oxime,but it is not efficient.In recent years,with the development of enzymology in biocatalysts,to design an efficient enzyme,which can detoxicate the poison of organic phosphorus in replacement of the use of chemical reagents has attracted more and more attention.The enzyme of DFPase is derived from the central nervous system of the Loligo vulgaris,and the PON1 is synthesized from the mammalian liver.Both of which can efficiently hydrolyze the organic phosphorus and make its P-F or P-O or P-CN bond broken.The similar functions and the delicate difference of the structures indicate that they may have an evolution relationship.Investigating the evolution of the catalysis mechanism may provide a theoretical basis for the study of structure-based protein engineering to develop a high performance antidote.In this paper,we study the catalysis mechanism of DFPase and PON1 through the density functional method B3 LYP.In the study of DFPase,we have designed two different nucleophilic attack pathways,which are the residue Asp229 and an activated water molecule as a nucleophile to attack on the phosphorus center of substrate respectively.The calculated results indicated that,both the Asp229 which coordinated with the Ca2+ and the H2 O which activated by the Asp229,are capable of proceeding nucleophilic attack on the substrate,and the reaction barriers are 14.8 kcal/mol and 6.0 kcal/mol,respectively.By contrast,the latter is more favorable reaction path way.And the Asp229 plays an important role whether it is as a nucleophile or a general base in the hydrolysis.Furthermore,the hexa-coordinated Ca2+ is the favorable conformation in the phosphoenzyme intermediate.In the whole hydrolysis reaction,residues Asn120 and Asn175 promote the elimination of the leaving-group via donating strong hydrogen bonds.Based on the study of catalysis mechanism of the DFPase,with H2 O as nucleophile,we have designed two PON1 models,the wild and the mutant models(Asn270 was mutated to H2 O to simulate the DFPase),then the substrate DFP and Paraoxon were combined with the two models to study the evolutionary relationship between PON1 and DFPase catalytic mechanism.The calculated results indicated that this mutation improved the activity of PON1 for substrate DFP and reduced the activity for substrate Paraoxon.Furthermore,this mutation also improved the stability of the PON1 towards substrate DFP.This paper not only explained the mechanisms of DFPase and PON1 to hydrolysis the organophosphorus,but also provided a new view of the evolutional mechanism of enzyme,which may do help to the synthesis of new drugs for detoxifying organophosphorus reagent.