Structural Network Analysis For Lipase Activity Based On Promotion Effect Of Dimethyl Sulfoxide Interaction

Selection of effective mutation site is highly critical in enzyme engineering,in particular for the sites distal from the active package,where no direct contact can be used between substrate and protein.In order to excavate long-distance regulation pathways inside functional protein and determine potential mutation sites,we choose an industrial enzyme as target protein,lipase,which has been widely used and studied so that the molecular mechanism is definitive up to date.To unveil the structural network,the promotion effect of dimethyl sulfoxide(DMSO)is tested in computation and experiment,to construct the amino acid pathways from the surface residues to the active sites.We developed a combined protocol for the structural topology analysis and kinetic experiments.First,a series of molecular dynamics simulations of lipase DMSO mixed systems show that the conformational diversity increases gradually with DMSO,and new types of conformations appears.Conformational clustering analysis illustrates the representative conformations in the systems at the different concentration of DMSO.The fluorescent experiment confirms the above conformational change,dependent on concentrations of the small molecule.In addition,kinetic analysis indicates that the catalytic efficiency is in a good correlation with concentrations of DMSO.The promotion effect of DMSO is also dependent of the structure of substrates,and the maximum reaction rate of V_m and Michaelis constant of K_m tend to the similar tendency.The structural topology analysis of amino acid network indicates that in the high concentration of DMSO the distance between the center of Lid and the catalytic activity decreases significantly.For the systems with the highest maximum reaction rate,the distance slightly rises,and the loop on colipase connection closes to Lid.It can be conclused from the statistical analysis that the paths between the loop and the lid become shorter owing to DMSO effect.Certain distal residues were proposed through the network analysis,providing a new insight for further parotein engineering.As a result,a software is prepared to solve such a problem,because solvent molecules cannot be included in currently available network softwares.In this work,structural bioinformatics,combined with traditional enzyme kinetic experiments,provides a feasible and basic stratagem to analyze distal effect and amino acid regulatory topological network.