Molecular Dynamics Simulations Study Of The Effects Of Several Different Factors On Enzyme Activity

Enzymes,as a class of biomolecules with high catalytic activity,facilitate the efficient performance of specific chemical reactions in the living body.As organisms are often in a dynamic and non-equilibrium state,protein molecular structures also show corresponding dynamic changes,and the conformational plasticity of proteins allows them to respond to changes in external environmental factors.The scientific community currently explores the structure-function relationships of proteins at the atomic level mainly through experimental techniques and protein structure prediction combined with deep learning.However,accurate measurement of dynamic changes in protein conformation caused by various factors is difficult due to spatial and temporal resolution limitations Thanks to the development of high-performance computers,molecular dynamics simulations have shown unique advantages in the study of protein structure-function and have become one of the important avenues in enzymology research.In this paper,the effects of several different factors(i.e.substrate binding,inhibitor binding,amino acid mutations,metal ion action and graphene oxide immobilisation)on the activity of their representative enzymes are investigated in depth using molecular dynamics simulations with the aid of enhanced sampling techniques.The specific research contents are as follows:1.The effect of substrate binding on the activity of glutamine 5'-phosphateribosylpyrophosphate aminotransferase(GPATase)The Glutamine 5'-phosphateribosylpyrophosphate aminotransferase(GPATase)links two structural domains(i.e.,glutaminase and phosphoribosyltransferase structural domains)through a 20 (?) NH3 channel.In this study,we combined conventional molecular dynamics(c MD)simulations and enhanced sampling accelerated molecular dynamics(a MD)simulations to characterize the coordinated catalytic mechanism of the two independent active structural domains in this enzyme.We concluded the following: Firstly,a comparison of protein conformational changes in GPATase+DON and GPATase+DON+c PRPP complexes showd that binding c PRPP to the flexible loop of PRTase(K326 to L350)had a strong influence on the formation of the R73-DON salt bridge;Secondly,only in the GPATase+DON+ c PRPP complex,the PRTase flexible loop can remain closed and had enough space to bind to c PRPP,indicating that the binding of DON to the glutamine loop had an effect on the conformational change of the PRTase flexible loop;Finally,both DON and c PRPP were tightly bound to the two domains,thus promoting the glutamine loop and the PRTase flexible loop to move close to each other and facilitating the the transfer of NH3.These theoretical results are useful for the study of efficient inhibitors related to GPATase.2.The effect of dipeptide inhibitor binding on the activity of angiotensinconverting enzyme(t ACE)Angiotensin-converting enzyme(ACE)-inhibiting peptides derived from food proteins lower blood pressure by inhibiting ACE activity.A recent study showed that although the dipeptides IY(Ile-Tyr)and LL(Leu-Leu)derived from soy protein had similar hydrophobicity and predicted activity values,the ACE inhibitory activity of IY was much higher than that of LL.Traditional experimental approaches were difficult to reveal the deep molecular mechanisms underlying this phenomenon.Therefore,1?s long Gaussian accelerated molecular dynamics(Ga MD)simulations were performed for Apo and two complex systems(ACE+LL and ACE+IY).The results showed that IY binding induced a significant contraction of the ACE activity pocket,mainly in the form of significant lateral shifts of ?13,?14,and ?15.In addition,hinge 2 and hinge 3were more stable compared to the ACE bound to LL.Moreover,during the simulation,?10 of ACE remained closed upon binding to IY.However,upon binding to LL,?10switched between the two open states.Taken together,our study provides detailed insights into inhibitor-induced conformational changes in ACE,which may help in the design of specific inhibitors targeting ACE for the treatment of hypertension.3.The effect of Q57H/W58P/D135 R mutation on phosphorylation activity of acid phosphatase(Pa APase)Ascorbic acid-2-phosphate(As A-2P)can be produced by efficient enzymatic synthesis method using cheap phosphate donors.The APase from Pseudomonas aeruginosa(Pa APase)can serve as a phosphorylation reaction enzyme.Since Pa APase is also a hydrolase,the phosphorylation efficiency is greatly affected.A recent report showed that Q6 mutations(Q57H/W58P/D135R)were able to increase the efficiency of phosphorylation by altering the local electric field.However,there is a lack of detailed explanation of how mutant residues improve the catalytic efficiency of phosphorylation by affecting protein conformational changes.Molecular dynamics simulations and protein network analysis showed that D135 R mutation can generate repulsive interaction with the key residue H132,which will induce proton transfer by the proximity of H132 to As A.Meanwhile,H132 can form a stable salt bridge interaction with E124,which will cause loop 7 to move inward and remain closed.Q57 H mutation facilitated shortening the communication paths between loop 4 and the catalytic domain.W58 P mutation may improve the disadvantage of blocking the binding of As A due to the large side chain size of tryptophan.Overall,we have characterized conformational changes in key regions caused by mutations,which may provide clues for enzyme design.4.The effect of metal ions on the activity of D-psicose 3-epimerase(CCH10DPEase)D-psicose is a rare sugar with low calorie and high sweetness,which can take an enzymatic reaction to convert D-fructose to D-psicose with DPEase.The activity of DPEase strictly depended on metal ions(Co2+,Mn2+),but the residual of heavy metal ions was not allowed in industrial production.Therefore,it was crucial to investigate the theoretical mechanism by which metal ions regulated the catalytic activity of DPEase for the design and modification of DPEase.In this study,we used molecular dynamics simulations(MD)and MM-PBSA free energy calculations to explore the differences in conformational changes and binding free energies of DPEase in the presence or absence of metal ions.Our theoretical results were as follows: Firstly,the two ?-helices D17-G29(?1),D121-G143(?4)in the complex without metal ions were unstable and partially unraveled compared to the DPEase+D-psicose complex containing heavy metals(Co2+,Mn2+);Secondly,during the simulations,the active pocket volume of the complexes without metal ions was larger than that with metal ions(Co2+,Mn2+);Thirdly,the reaction channel characteristics were also significantly different,with wider and longer reaction channels in the metal ion-free than in the metal ion-containing system;Finally,the free energy of PPEase binding to the substrate was lower in the complexes containing metal ions(Co2+,Mn2+).This indicated that the substrate binding was more stable in the presence of metal ions.In summary,the ordered structure around the active residues,smaller pockets and narrower channels will provide a stable environment for the racemization reaction to occur.Our theoretical results will contribute to the further study of DPEase.5.The effect of graphene oxide immobilization on the thermal stability of Dpsicose 3-epimerase(agtu DPEase)Thermal stability is a limiting factor for effective application of D-psicose 3-epimerase(DPEase)enzyme.Recently,it was reported that the thermal stability of DPEase was improved by immobilizing enzymes on graphene oxide(GO)nanoparticles.However,the detailed mechanism is not known.In this study,we investigated interaction details between GO and DPEase by performing molecular dynamics(MD)simulations.The results indicated that the domain(K248 to D268)of DPEase was an important anchor for immobilizing DPEase on GO surface.Moreover,the strong interactions between DPEase and GO can prevent loop ?1-?1 and ?4-?4 of DPEase from the drastic fluctuation.Since these two loops contained active site residues,the geometry of the active pocket of the enzyme remained stable at high temperature after the DPEase was immobilized by GO,which facilitated efficient catalytic activity of the enzyme.Our research provided a detailed mechanism for the interaction between GO and DPEase at the nano biology interface.