Catalytic Inactivation Of Pseudomonas Aeruginosa Arylsulfatase During Reaction And Its Mechanism

To obtain the label enzyme for enzyme-linkedimmunoabsorbent-assay of two components each time in one well with conventional microplate readers,and new labeling enzyme for chemiluminescent,molecular engineering of pseudomonas aeruginosa arylsulfatase(PAAS)is needed.During molecular engineering and screening,it was found that PAAS and its site-directed mutants in a reaction system with p-nitrophenol sulfate as the substrate but no any inhibitor at the optimum reaction pH and temperature,there was a significant reduction of activity during the catalytic reaction with the insufficient accumulation of product,referring to catalytic inactivation of enzyme.Through caracterizations of kinetics,mitigating effects of polyclonal antibody and special small peptide,and structural analysis,a mechanism of double activity sites was inferred,and thereby a kinetic model of catalytic inactivation was proposed.1.Characterization of inactivation for PAAS and its mutants1)Under the same reaction condition,the reaction curves of PAAS and mutants at different substrate concentrations of p-nitrophenyl sulfate(4PNS)were continuously monitored.Kinetic curves of PAAS and most of its mutants were obviously bent with the extension of the reaction time,this phenomenon is definited as catalytic inactivation which was related to the concentrations of the catalyzed substrate.M72 K is the mutant of the most obvious inactivation;the enzymes before and after the catalytic reactionwas analyzed by ultra high-resolution mass spectrometry,and covalently sulfation modification on amino acid residues was found.;2)The polyclonal antibody against PAAS and the synthesized polypeptide containing the modified amino acid fragment were added to the reaction system of M72 K,to detect the effect on the inactivation of the mutant M72 K.It was found that the bending degree of the reaction curves of M72 K were significantly relieved;2.kinetic model for the inactivation of site-directed mutant M72K1)Bseong on the classical two-step reaction catalyzed by sulfatasea classical model was first proposed,but it could not simulate the accumulation of product and sulfatated enzyme intermediates.Therefore,a new model for inactivation relating to doubleactive site kinetic was proposed though iterative numerical integration with sets of parameter,which fit well the inactivation curves of M72 K and simulated the accumulation process of intermediates.2)The accumulation of enzyme intermediates obtained by the King-Altman method is consistent with the results obtained from this new model,so that the rationality of the model results is proved.3.The application of catalytic inactivation modelThrough inactivation kinetic model,alivation of the inactivation of M72 K by the peptides and anti-PAAS polyclonal antibody were fit to elabrate the inactivation mechanism during enzyme reaction.In summary,the inactivation of PAAS and its mutants during the reaction is related to the sulfation of the enzymes.This inactivation can be effectively alleviated by anti-PAAS polyclonal antibodies and specific peptides;The dual-activity centers inactivation kinetic model was propose,which gave the results of the accumulation of product and the intermediates consistent with those of the classical King-Altman analysis method.The rationality of the inactivation mechanism was preliminarily proved,to provide a theoretical basis for the discovery of high activity,good stability of mutants through molecular engineering of PAAS.