| Enzymes are a kind of complex biomacromolecules with the biocatalytic function,which play an indispensable role in the living world.An understanding of the enzymatic processes in nature can not only help us to unravel the mystery of biocatalysis,but also provide inspiration for design of high-performance biomimetic catalysts.The whole process of enzymatic catalysis generally includes four chemical and non-chemical steps:transportation of the substrate into the active site,binding of the substrate at the active site,triggering of the catalytic reaction and releasing of the product from the active pocket.Accordingly,it is highly required for zymochemistry to understand the catalytic mechanism of chemical steps and the channel mechanism of non-chemical steps.Here molecular dynamics(MD)simulations and combined quantum mechanics and molecular mechanics molecular dynamics(QM/MM MD)simulations with umbrella sampling were used to explore the catalytic.A detailed study of the catalytic reaction and channel mechanism in the enzymatic catalysis was carried out to reveal the thermodynamical and dynamical properties for the substrate delivery and the chemical reaction,as well as the rate-determining step and the effect of key residues.The main research contents and results of this dissertation are summarized as follows:(1)Extensive QM/MM MD and MM MD simulations have been used to explore the whole journey of the enzymatic detoxification for the G-type nerve agent sarin by PTE,including the substrate delivery to the active site,the catalytic reaction,and the release of products.Here both divalent zinc ions(Zn? and Zn?)in the active site are ligated by the carboxylate group(Lys169),the nucleophile bridging hydroxide ion,and two water molecules,resulting in the six-coordinated metal centers in the ready state of PTE.RAMD-MD simulations reveal that the channel Pa,along the leaving pocket,is basically responsible for the substrate delivery to the active site with an energy release of 16.8 kcal/mol.The enzymatic P-F cleavage of the substrate sarin follows a two-step mechanism,in which the nucleophilic attack of the bridging hydroxide leads to the P-O? formation with the free-energy barrier of 9.8 kcal/mol,and followed by the P-F cleavage and the proton transfer from the ?-OH to the residue Asp301,the degraded product is formed with the free-energy span of 12.3 kcal/mol.After the F-release,the degraded product is tightly bound to the binuclear zinc center in the bidentate coordination,and its dissociation is predicted to be rate-determining for the enzymatic efficiency.The multiple chemical steps are involved in dissociation of the degraded product,and the predicted free-energy barriers for the low-energy pathway are 21.0 kcal/mol for the recombination step and 18.3 kcal/mol for dissociation of the neutral product.The solvation of products in the bulk solvent is generally exothermic remarkably,which may drive the release of products.The present work provides a comprehensive understanding of the enzymatic detoxification of sarin by PTE,which is important for further experimental studies and the enzyme engineering of degrading toxic organophosphorus compounds.(2)The pyrimidine-specific nucleoside hydrolase Yeik(CU-NH)from Escherichia coli cleaves the N-glycosidic bond of uridine and cytidine with a 102-104fold faster rate than that of purine nucleoside substrates,such as inosine.Such remarkable substrate specificity and the plausible hydrolytic mechanisms of uridine have been explored by using QM/MM and MM MD simulations.The structural features of active domain,loop motion,substrate specificity,substrate delivery,and plausible catalytic mechanisms have been discussed.Current calculations and simulations indicate that the anti-uridine substrate has stronger binding interactions with the activesite residues,compared to the syn-uridine,and as a favorable configuration,the antiuridine is involved in the enzymatic hydrolysis.Once the substrate is bound to the active site,the flexible loop1 and loop2,as well as the protein backbone become more ordered,and the hydrogen-bond interactions between the substrate and the side-chain residues of loops may contribute the substrate specificity to some extent,although it is basically dominated by the kinetic barriers.The access of uridine to the active site is predicted to be much more favorable thermodynamically,compared to the inosine substrate.Here,the enzymatic cleavage of the N-glycosidic bond by CU-NH follows a highly dissociative concerted and nonsynchronous mechanism,and no activity enhancement from the substrate protonation has been found.The present results provide new insights into the enzymatic hydrolysis by pyrimidine nucleoside hydrolases.(3)Naphthalimide derivatives are types of small-molecule anticancer drug candidates.However their negative factors and potential side effects make their application limited.The pharmacophores select a direct access into the tumor cells as the first choice and can reduce the side effect of the anti-cancer drugs on the normal cells.Herein,the delivery and binding of the naphthalimide-polyamine complex assisted by the bovine serum albumin(BSA)protein have been studied by combining several molecular dynamics simulations.The plausible transportation channels for the delivery of the naphthalimide-polyamine complex to two drug sites(DSI and DSII),and corresponding thermodynamic and dynamic properties and the mechanisms have been discussed in detail.The binding mode,binding energy and substituent effects have been also identified.The two drug sites have different preferences towards the compound with the electron-withdrawing and electron-donating substituents,and their strong interactions are more sensitive to the number of the substituent groups.This selective specificity of these two drug sites manipulated by the electron-withdrawing and electron-donating substituents is quite promising for the design of new naphthalimide drugs,and the site-selectivity-dependence design strategy may open up an avenue in drug discovery. |
PDF Full Text Request