Mechanism Of Prolyl Endopeptidases-catalyzed Degradation Of Gluten

Celiac disease is a chronic intestinal disease.The ingested gluten protein by patients cannot be hydrolyzed properly leading to the autoimmune-related diseases.The proportion of celiac disease patients is as high as one percent in Western countries.Gluten protein is one of the main sources of plant protein in daily life,widely found in wheat,barley and other food crops.At present,eating gluten-free foods for life is regarded as the only effective way to treat celiac disease for patients.However,because of cumbersome processes,high price,bad taste and nutrients deficiency,patients have an expectation of a new treatment which they can have a normal eating without causing intestinal disease.The new treatment is the starting point of this research.This thesis mainly includes three chapters,which are described as follows:Summarizes what is celiac disease and its pathogenesis,and the source and composition of gluten protein.This chapter also introduces the current treatment of celiac disease,including gluten-free food therapy,grain modification,cutting off the passage of gluten protein into the small intestinal epithelial cells,immunotherapy,vaccines and gluten protein degrading enzyme therapy.Based on the gluten protein degrading enzyme therapy,we make a further introduction on Proline endopeptidase’s structure,substrate specificity and other related characteristics.We make a Molecular Dynamics simulation on the process of the substrate pNA and peptide PQPQLPYPQPQLP enter the open or closed state MX PEP enzyme(Myxococcus xanthus)and SC PEP enzyme(Sphingomonas capsulata)by using the Molecular Dynamics method.The results show that: when it is in the absence of a substrate,the MX PEP enzyme tends to be closed;when there are some substrate molecules,the MX PEP enzyme turns to the open state;when there are two pNA molecules,one of the substrates Molecules can enter the enzyme pocket.Besides,the results also show that: when there is no substrate,the SC PEP enzyme tends to be turned on;when there are some substrate molecules,the substrate molecules can enter the enzyme pocket;when the substrate is a peptide molecule and it enters the SC PEP enzyme pocket,the substrate can induce the shutdown of the SC PEP enzyme.The results of the study indicate that the substrate molecule is an important factor in inducing the opening of the PEP enzyme and when the PEP enzyme exists,the substrate molecule will spontaneously move into the enzyme pocket for catalytically degradation.We build a 3D Model which is based on the structure of analyzed MX PEP enzyme and SC PEP enzyme to study the mechanism of PEP enzyme catalyzing the degrading the gluten.By using QM / MM(Quantum Mechanics/Molecular Mechanics)calculations,the mechanisms of these two enzymes’ degradation on substrate molecule pNA are studied.Calculations show that the degradation of substrate molecules by MX PEP enzyme is divided into two stages,namely acylation and deacylation stages.A single-step reaction pathway and two-step reaction pathway are two competing approaches in the acylation stage.The single-step reaction pathway is Oγ’s nucleophiles on the side chain of Ser533 attacking the carbonyl group of the substrate,and the proton of Hγ is transferred simultaneously.The two-step reaction pathway is Oγ’s nucleophiles on the side chain of Ser533 attacking the carbonyl group of the substrate and then obtaining the stable intermediate INT1 b,and completes transferring the proton of Hγ.The free energy barriers of the two pathways individually are 39.3 and 21.4kcal/mol.The deacylation stage is a single one-step reaction pathway,and there was a water molecule close to the side chain of Ser533 that was acylated.Subsequently,the oxygen atom of the water molecule attacks the acyl group attached to Ser533,and is transferred through the protons of water molecules.The free energy barrier for this step is 25.0 kcal/mol.The results of the study indicate that the vital and accelerate step of deacylation of the enzyme is the reaction that MX PEP enzyme catalyzes the degradation of the substrate molecule pNA.The SC PEP enzyme is composed of acylation and deacylation stages in the degradation of substrate molecules.The acylation stage is a single single-step reaction pathway.The nucleophilic offensive substrate carbonyl group and proton transfer of the side chain of Ser575 are completed at the same time;the free energy barrier of the reaction is 20.7 kcal/mol.The deacylation stage is a single two-step reaction pathway.After the nucleophilic attack of the oxygen atom in the water molecule is completed,the stable intermediate INT2 is obtained,and then the proton transfer on the water is completed;the free energy barrier of the reaction is 22.8 kcal/mol.The calculation results show that the rate-determining step of the reaction that SC PEP enzyme catalyzes the degradation of the substrate molecule pNA is the deacylation stage also.Based on the above studies,we found that the rate-determining step for PEP enzymes to catalyze the hydrolysis of peptides should be the reaction of catalyzing the deacylation of serine in the triad.The research on the reaction of PEP enzyme to catalyze the degradation of gluten and the study of the Molecular Dynamics simulation structure of the substrate in this article reveal the unique mechanism of the reaction catalyzed by PEP enzyme.These studies will provide theoretical support for the later development of new drugs for celiac disease and the redesign of PEP enzymes.