Study On The Effect Mechanism Of Microenvironment Regulation On Enzyme Activity

Enzyme catalysis plays a vital role in life activities such as metabolic processes.The catalytic process of enzymes in nature is mostly realized by multiple steps or cascade reactions.As far as the multi-enzyme catalytic system is concerned,the catalytic activity of each enzyme should be fully utilized to solve the incompatibility of the cascade reaction through microenvironmental engineering,and the catalytic activity of the enzyme should be adjusted,so as to achieve multi-enzyme cooperative and efficient catalysis.In this paper,charged polymers are used to covalently modify enzymes to create an acid/base microenvironment.The degree of modification is optimized and the mechanism is explained by molecular dynamics simulations.The improvement of enzyme catalytic activity is verified by experiments.The main work of this paper is as follows:(1)Microenvironment system catalyzed by dual enzymes was constructed.The protein states of cytochrome C(Cyt C)and D-amino acid oxidase(DAAO)at five pH values of 2,4.5,7,8.5,and 10 were calculated by H++,and the charge distribution was calculated by Gauss for GROMACS simulation.Solvent accessibility analysis was used to determine the modification site of the dual-enzyme protein.Cyt C modified five repeat units of polyacrylic acid(PAA)at three different degrees at five different pH values.DAAO modified five repeat units of polyallylamine(PAM)at four different degrees at five different pH values.(2)The influence mechanism of microenvironment regulation on the dual enzyme system was analyzed.By reading the trajectory files in the range of 100 ns of a total of 45 systems with dual enzymes at different pH values and different degrees of modification,the analysis of net charge,electrostatic potential energy,solvent-accessible surface area(SASA)and the number of hydrogen bonds between proteins and water shows gradient increase,indicating the modification increases the charge and polarity of the enzyme-polymer complex;the analysis of root-mean-square deviations(RMSD),hydrogen bonds in protein molecules and secondary structure proved that charged polymers can buffer the influence of extreme pH value on the stability of enzymes while maintaining the spatial structure of proteins,and realized the protection of microenvironment.In addition,the optimal modification degrees of the dual enzymes were determined to be[Cyt C-PAA]0.7 and[DAAO-PAM]0.7,respectively,and the active site and thermal stability were analyzed.(3)The regulation of polymer microenvironment on enzyme activity was verified.Cyt C and DAAO covalently modified PAA with a molecular weight of 5000 Da and PAM with a molecular weight of 17,000 Da by the EDC-NHS method,respectively.While increasing the particle size and charge of the enzyme protein,the integrity of the protein structure was retained.After the double enzyme modification,the enzyme activity showed different degrees of improvement at different pH values.At pH=7,the enzyme activity of the modified Cyt C increased by 2.2-fold;at pH=2 and pH=4.5,the enzyme activity of the modified DAAO increased by 2.1-fold.In addition,under the optimal pH value,the enzymatic activity of the dual enzymes after covalent modification of the polymer was also significantly improved.