Theoretical Studies On The Catalytic Mechanisms Of Three Desmoenzymes

As the foundation of the metabolism of organisms,enzymes are the most common biological catalyst in nature.Compared with ordinary catalysts,enzymes have some distinct characteristics,such as a high catalytic efficiency,mild reaction conditions and high degree of specificity.With the increasingly emerging of new technology,the theory and application of enzyme also made great development and has become the core of industrial biotechnology.In recent years,the research about enzyme catalysis and renovation is becoming a hot topic in biocatalysis field through simulating the biological macromolecule by the computer technology,which helps people understand the relationship between catalytic function and structure of enzymes.In this paper,based on the latest crystal structure in experiment,catalytic mechanisms of three kinds of enzymesare were studied by the combination of quantum mechanics and molecular mechanics(QM/MM)method.Our calculation results forecast the structures of reactants,transition states,intermediates and products,and give the reaction energy barriers,and illustrate the best reaction pathway and the rate-limiting step of reaction,and describe the reaction details at the atomic level.The results not only explain the experiment phenomenon,but also predict the roles of some key residues,which makes up for the deficiency of the experimental research.In addition,the research results provide a reference for the studies of catalytic mechanism of related enzymes.The main research contents are as follows:(1)Mechanism of the Glutathione Persulfide Oxidation Process Catalyzed by Ethylmalonic Encephalopathy Protein 1.Ethylmalonic encephalopathy protein 1(ETHE1)is a ?-lactamase fold-containing protein,which is non-heme iron-dependent oxygenase and related with the increased cellular levels of hydrogen sulfide.ETHE1 is essential for the survival of a range of organisms and catalyzes the oxidation of glutathione persulfide(GSSH).Currently,the catalytic mechanism of ETHE1 still remains unclear,despite a catalytic cycle has been suggested from the crystal structure and a proposal for the mechanistically related cysteine dioxygenase(CDO).We performed a series of QM/MM calculations on the substrate GSSH oxidation by human ETHE1.Our calculation results reveal that the ground state of theiron(?)-superoxo reactant is quintet,which can be described as GSS+·-Fe(?)-O2· and the most feasible reaction channel was found to starts from the cleavage of dioxygen and a concerted attack of distal oxygen on the sulfur atom of substrate,forming the metal-bound activated oxygen and a sulfite-intermediate.Moreover,the reaction starts from quintet ground state reactant,undergoes triplet intermediate and finally generates the septet product rather than the reaction of CDO which starts from a singlet-quintet crossing.(2)Theoretical Study of the Hydrolysis Mechanism of Dihydrocoumarin Catalyzed by Serum Paraogonase 1(PON1).Serum paraoxonasel(PON1)is a calcium-dependent enzyme that can catalyze the hydrolysis of multiple substrates,including the lactones,thiolactones,carbonates,esters and phosphotriesters,as well as the formation of a variety of lactones.To better understand the lactonase mechanism of PON1,the hydrolysis of dihydrocoumarin:which is considered as one kind of native substrate of PON1,has been investigate by using QM/MM method.Two possible reaction pathways with either Glu53 or His115 acts as the general base have been considered,which correspond to the overall energy barriers of 12.5 and 6.9 kcal/mol,respectively.During the catalytic reaction,if one of the two residues(Glu53 and His 115)acts as the catalytic base,the other one forms strong hydrogen bonding interaction with the attacking hydroxide to facilitate the reaction.However,mutation studies also reveal that Glu53 is necessary for hydrolysis,whereas His115 is not essential but can promote the activity of PON1.Natural population analysis indicates that the catalytic Ca2+ does not act as Lewis acid but mainly plays role in fixing the orientations of the substrate and related residues.In addition,Asp269 is found to coordinate with Ca2+ cation and facilitate the protonation of the alkoxide leaving group by forming hydrogen bond with lactone.These results can explain the fact that mutation of Glu53 results in the loss of activity of PON1,and the hydrolysis of dihydrocoumarin is unaffected by mutation of H115.(3)Theoretical study of the y-elimination mechanism of the L-methionine catalyzed by Methionine ?-lyase(MGL).Methionine ?-lyase(MGL)is a pyridoxal 5'-phosphate(PLP)-dependent enzyme,catalyzing the ?-elimination reaction of L-methionine.The ?-elimination mechanism of the L-methionine catalyzed by methionine ?-lyase was studied by the QM/MM approach.According to the calculated results,the whole mechanism is divided into two parts.Part I describes the formation process of external aldimine intermediate from internal aldimine intermediate,in which Lys210 leaves off with the assistance of a water molecule with energy barrier of 14.6 kcal/mol.In Part ?,the reaction mechanism from external aldimine intermediate to aminocrotonate intermediate was explored,in which the Ca-proton of substrate methionine transfers to C4' atom of PLP by Lys210.And the C?-proton transfers to S atom of the methionine by Try113 and simultaneously the thiol group eliminates to generate unsaturated ketimine intermediate.In the end,the C4'-proton transfers to the terminal C atom of unsaturated ketimine to generate the aminocrotonate intermediate.The rate-limiting step of the whole reaction might be the abstraction of Ca-proton and the transfer of the C4'-proton by Lys210 with 21.1 kcal/mol and 21.5 kcal/mol,respectively.These results can provide useful information for the mechanism research of PLP-dependent lyase.