3D-QSAR and molecular docking studies of peptide-hybrids as dengue virus NS2B/NS3 protease inhibitors.

Global warming and climate change have made dengue disease a global health issue. More than 50 % of the world's population is at danger of dengue virus (DENV) infection, according to the World Health Organization (WHO). Therefore, a clinically approved dengue fever vaccination and effective treatment are needed. Peptide medication development is new pharmaceutical research. Here we intend to recognize the structural features inhibiting the DENV NS2B/NS3 serine protease for a series of peptide-hybrid inhibitors (R1-R2-Lys-R3-NH2) by the 3D-QSAR technique. Comparative molecular field analysis (q2 = 0.613, r2 = 0.938, r2pred = 0.820) and comparative molecular similarity indices analysis (q2 = 0.640, r2 = 0.928, r2pred = 0.693) were established, revealing minor, electropositive, H-bond acceptor groups at the R1 position, minor, electropositive, H-bond donor groups at the R2 position, and bulky, hydrophobic groups at the R3 position for higher inhibitory activity. Docking studies revealed extensive H-bond and hydrophobic interactions in the binding of tripeptide analogues to the NS2B/NS3 protease. This study provides an insight into the key structural features for the design of peptide-based inhibitors of DENV NS2B/NS3 protease.

Mechanism of cationic ring-opening polymerisation of ε-caprolactone using metallocene/borate catalytic systems: a DFT and NCI study on chain initiation, propagation and termination.

We present a comprehensive DFT investigation on the cationic ring-opening polymerisation (CROP) of ε-caprolactone (CL) using zirconocene/borate catalyst systems. All possible pathways of the interaction between cationic species [Cp2ZrMe+] and counteranions, [A-] = [MeB(C6F5)3]- and [B(C6F5)4]-, were examined during chain initiation, propagation, and termination steps. The calculations reveal an active chain-end mechanism with O-alkyl bond cleavage of the polymerisation. The catalytic performance of the two counteranions is found to be identical, and they influence the initial process through stabilisation of the cationic species via non-covalent interactions (NCI), with the [MeB(C6F5)3]- anion stabilising the catalyst-monomer complex more effectively than the [B(C6F5)4]- anion by 24.3 kJ mol-1. The first two propagations are likely the rate-determining step, with calculated free-energy barriers of 61.4-71.2 and 73.9-80.6 kJ mol-1 with and without the anions (A-), respectively. The presence of the counteranion significantly affects the third propagation rate, lowering the barriers up to 20 kJ mol-1. Comparison of the first termination and the third propagation shows that they are not competitive, with the termination being less facile. We also studied the initiation and propagation steps for the hafnocene catalyst and found that the Hf catalyst slightly favours the CL CROP in comparison to the Zr catalyst. Analysis of solvent and dispersion interaction demonstrates that both factors play an important role in the process. NCI analysis reveals weak (van der Waals) interactions at the contacts between the cationic species and the counteranions during the reaction course. Overall, our results offer insights into the structures and interactions involved in the polymerisation.

Binding interactions and in silico ADME prediction of isoconessimine derivatives as potent acetylcholinesterase inhibitors.

In pursuit of new acetylcholinesterase (AChE) inhibitors for treating Alzheimer's disease (AD), a series of ten previously synthesized isoconessimine compounds (7a-7j) was in silico investigated for their binding interactions with AChE and pharmacokinetics based on absorption, distribution, metabolism, and excretion (ADME) properties using molecular docking, ONIOM (Our own N-layered Integrated molecular Orbital and molecular Mechanics) method and SwissADME tools. Docking experiments showed that all compounds bind within the active site gorge of AChE (PDB entry 1C2B), posing its aryloxy-substitutional ethyl group to catalytic site and conessine skeleton to peripheral anionic site. ONIOM interaction energy was used as an ONIOM score to improve docking score, and it ranked 7b as the most potent AChE inhibitor, in agreement with previous experiment. Residues, ASP74, TRP86, GLY122, GLU202, TRP286, GLU292, SER293, ILE294, TYR337, TYR341, and HIS447 were identified as important for the binding of the AChE-isoconessimine complex. The SwissADME investigation suggested that four compounds (7a, 7c, 7d and 7f) agree with the rules of drug-likeness. The steric and electronic effects on the aryloxy-substitutional ethyl group as important factors in the AChE inhibition were also discussed, which brings a better understanding of Alzheimer's disease drug development.

Computational evaluation of zirconocene catalysts for ε-caprolactone cationic ring-opening polymerization.

This quantum chemical study presents the ligand effect and a structure-property relationship in the cationic ring-opening polymerization (CROP) of ε-caprolactone using zirconocene catalysts. We first examined the effects of catalyst structure on the initiation and chain propagation steps of the CROP process. A total of 54 catalyst structures were investigated to understand the influence of the ligand structure on the stability of the catalyst-monomer complex and polymerization activity. The properties of the catalysts were analyzed in terms of ancillary ligands, ligand substituents, and bridging units. Calculations showed that the polymerization follows a proposed cationic mechanism, with ring opening occurring via alkyl-bond cleavage. A correlation between complex stability and activation energy was also observed, with ligand substituents dominating in both steps. While the ancillary ligands directly affect the HOMO energy level, the bridges are mainly responsible for the catalyst geometries, resulting in reduced complex stability and higher activation energy for the propagation step. This study contributes to a better understanding of the structural characteristics of zirconocene catalysts, which offers guidance for improving CROP activities in lactone polymerization.

Structural basis for inhibition of a GH116 ß-glucosidase and its missense mutants by GBA2 inhibitors: Crystallographic and quantum chemical study.

The crystal structure of the Thermoanaerobacterium xylanolyticum in glycoside hydrolase family 116 (TxGH116) ß-glucosidase provides a structural model for human GBA2 glucosylceramidase, an enzyme defective in hereditary spastic paraplegia and a potential therapeutic target for treating Gaucher disease. To assess the therapeutic potential of known inhibitors, the X-ray structure of TxGH116 in complex with isofagomine (IFG) was determined at 2.0 Å resolution and showed the IFG bound in a relaxed chair conformation. The binding of IFG and 7 other iminosugar inhibitors to wild-type and mutant enzymes (Asp508His and Arg786His) mimicking GBA2 pathogenic variants was then evaluated computationally by two-layered ONIOM calculations (at the B3LYP:PM7 level). Calculations showed that six charged residues, Glu441, Asp452, His507, Asp593, Glu777, and Arg786 influence inhibitor binding most. His507, Glu777 and Arg786, form strong hydrogen bonds with the inhibitors (∼1.4-1.6 Å). Thus, the missense mutation of one of these residues in Arg786His has a greater effect on the interaction energies for all inhibitors compared to Asp508His. In line with the experimental data for the inhibitors that have been tested, the favorable interaction energy between the inhibitors and the TxGH116 protein followed the trend: isofagomine > 1-deoxynojirimycin > glucoimidazole > N-butyl-deoxynojirimycin ≈ N-nonyl-deoxynojirimycin > conduritol B epoxide ≈ azepane 1 > azepane 2. The obtained structural and energetic properties and comparison to the GBA2 model can lead to understanding of structural requirement for inhibitor binding in GH116 to aid the design of high potency GBA2 inhibitors.

Multiscale QM/MM Simulations Identify the Roles of Asp239 and 1-OH···Nucleophile in Transition State Stabilization in Arabidopsis thaliana Cell-Wall Invertase 1.

Arabidopsis thaliana cell-wall invertase 1 (AtCWIN1), a key enzyme in sucrose metabolism in plants, catalyzes the hydrolysis of sucrose into fructose and glucose. AtCWIN1 belongs to the glycoside hydrolase GH-J clan, where two carboxylate residues (Asp23 and Glu203 in AtCWIN1) are well documented as a nucleophile and an acid/base catalyst. However, details at the atomic level about the role of neighboring residues and enzyme-substrate interactions during catalysis are not fully understood. Here, quantum mechanical/molecular mechanical (QM/MM) free-energy simulations were carried out to clarify the origin of the observed decreased rates in Asp239Ala, Asp239Asn, and Asp239Phe in AtCWIN1 compared to the wild type and delineate the role of Asp239 in catalysis. The glycosylation and deglycosylation steps were considered in both wild type and mutants. Deglycosylation is predicted to be the rate-determining step in the reaction, with a calculated overall free-energy barrier of 15.9 kcal/mol, consistent with the experimental barrier (15.3 kcal/mol). During the reaction, the -1 furanosyl ring underwent a conformational change corresponding to 3E ↔ [E2]⧧ ↔ 1E according to the nomenclature of saccharide structures along the full catalytic reaction. Asp239 was found to stabilize not only the transition state but also the fructosyl-enzyme intermediate, which explains findings from previous structural and mutagenesis experiments. The 1-OH···nucleophile interaction has been found to provide an important contribution to the transition state stabilization, with a contribution of ∼7 kcal/mol, and affected glycosylation more significantly than deglycosylation. This study provides molecular insights that improve the current understanding of sucrose binding and hydrolysis in members of clan GH-J, which may benefit protein engineering research. Finally, a rationale on the sucrose inhibitor configuration in chicory 1-FEH IIa, proposed a long time ago in the literature, is also provided based on the QM/MM calculations.

A computational study of the reaction mechanism and stereospecificity of dihydropyrimidinase.

Dihydropyrimidinase (DHPase) is a key enzyme in the pyrimidine pathway, the catabolic route for synthesis of ß-amino acids. It catalyses the reversible conversion of 5,6-dihydrouracil (DHU) or 5,6-dihydrothymine (DHT) to the corresponding N-carbamoyl-ß-amino acids. This enzyme has the potential to be used as a tool in the production of ß-amino acids. Here, the reaction mechanism and origin of stereospecificity of DHPases from Saccharomyces kluyveri and Sinorhizobium meliloti CECT4114 were investigated and compared using a quantum mechanical cluster approach based on density functional theory. Two models of the enzyme active site were designed from the X-ray crystal structure of the native enzyme: a small cluster to characterize the mechanism and the stationary points and a large model to probe the stereospecificity and the role of stereo-gate-loop (SGL) residues. It is shown that a hydroxide ion first performs a nucleophilic attack on the substrate, followed by the abstraction of a proton by Asp358, which occurs concertedly with protonation of the ring nitrogen by the same residue. For the DHT substrate, the enzyme displays a preference for the L-configuration, in good agreement with experimental observation. Comparison of the reaction energetics of the two models reveals the importance of SGL residues in the stereospecificity of catalysis. The role of the conserved Tyr172 residue in transition-state stabilization is confirmed as the Tyr172Phe mutation increases the activation barrier of the reaction by ∼8 kcal mol-1. A detailed understanding of the catalytic mechanism of the enzyme could offer insight for engineering in order to enhance its activity and substrate scope.

Role of water coordination at zinc binding site and its catalytic pathway of dizinc creatininase: insights from quantum cluster approach.

Creatininase is a key enzyme of creatinine-metabolizing pathway in mammals, and has a great potential for diagnostic application. It catalyzes the reversible conversion of creatinine to creatine. Here, we investigated its reaction mechanism with density functional theory in conjunction with the quantum cluster approach. Three reaction pathways in which several possible proton transfers assisted by either His178 or a water ligand to Zn1 (Wat2) or both were considered. DFT calculations reveal, depending on Wat2 coordination mode at Zn1, two competitive ring-opening pathways where His178 playing a central role as a proton shuttle or both His178 and Wat2 serving as a dual catalytic role as a base and an acid, respectively. Three elementary steps were proposed for the reaction: the first involves nucleophilic attack by a bridging hydroxide to the substrate and forms a gem-diolate intermediate, followed by a proton transfer from the gem-diolate to His178 (His178 protonation is a required step for efficient proton transfers). Finally, the second proton transfer from the protonated His178 or Wat2 to the amide of substrate leads to the ring opening. The first proton transfer is the rate-limiting step of the whole reaction, in consistent with previous experimental and computational studies. A detailed understanding of the reaction mechanism of the creatininase enzyme family will also be helpful for developing a biosensor for kidney function.

Active site dynamics and catalytic mechanism in arabinan hydrolysis catalyzed by GH43 endo-arabinanase from QM/MM molecular dynamics simulation and potential energy surface.

The endo-1,5-α-L-arabinanases, belonging to glycoside hydrolase family 43 (GH43), catalyse the hydrolysis of α-1,5-arabinofuranosidic bonds in arabinose-containing polysaccharides. These enzymes are proposed targets for industrial and medical applications. Here, molecular dynamics (MD), potential energy surface and free energy (potential of mean force) simulations are undertaken using hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to understand the active site dynamics, catalytic mechanism and the electrostatic influence of active site residues of the GH43 endo-arabinanase from G. stearothermophilus. The calculated results give support to the single-displacement mechanism proposed for the inverting GH43 enzymes: first a proton is transferred from the general acid E201 to the substrate, followed by a nucleophilic attack by water, activated by the general base D27, on the anomer carbon. A conformational change (2E ↔E3 ↔ 4E) in the -1 sugar ring is observed involving a transition state featuring an oxocarbenium ion character. Residues D87, K106, H271 are highlighted as potential targets for future mutation experiments in order to increase the efficiency of the reaction. To our knowledge, this is the first QM/MM study providing molecular insights into the glycosidic bond hydrolysis of a furanoside substrate by an inverting GH in solution.Communicated by Ramaswamy H. Sarma.

Data on electronic structures for the study of ligand effects on the zirconocene-mediated trimethylene carbonate polymerization.

The data presented in this paper are related to the research article entitled "Effect of ligand structure in the trimethylene carbonate polymerization by cationic zirconocene catalysts: A "naked model" DFT study" (Jitonnom and Meelua, 2017) [1]. In this data article, we present 3D molecular information of 29 zirconocene catalysts that differ in electronic and steric properties. The data contains all cationic species along the initiation and first propagation step of the polymerization, which are provided in a PDB format that can be used for further studies.

Data characterizing the energetics of enzyme-catalyzed hydrolysis and transglycosylation reactions by DFT cluster model calculations.

The data presented in this paper are related to the research article entitled "QM/MM modeling of the hydrolysis and transfructosylation reactions of fructosyltransferase from Aspergillus japonicas, an enzyme that produces prebiotic fructooligosaccharide" (Jitonnom et al., 2018) [1]. This paper presents the procedure and data for characterizing the whole relative energy profiles of hydrolysis and transglycosylation reactions whose elementary steps differ in chemical composition. The data also reflects the choices of the QM cluster model, the functional/basis set method and the equations in determining the reaction energetics.

QM/MM modeling of the hydrolysis and transfructosylation reactions of fructosyltransferase from Aspergillus japonicas, an enzyme that produces prebiotic fructooligosaccharide.

Fructosyltransferases (FTs) act on sucrose by cleaving the ß-(2→1) linkage, releasing glucose, and then transferring the fructosyl group to an acceptor molecule. These enzymes are capable of producing prebiotic fructooligosaccharides (FOSs) that are of industrial interest. While several FOS-synthesizing enzymes FTs have been investigated, their catalytic mechanism is not yet fully understood, especially the molecular details of how FOS are enzymatically synthesized from sucrose. Here, we present a comparative quantum mechanics/molecular mechanics (QM/MM) study on the hydrolysis and transfructosylation reactions catalyzed by A. japonicus FT using sucrose as donor and acceptor substrates. It is shown that the hydrolysis and transfructosylation reactions of the enzyme seem to be competitive with similar potential energy profiles. For all studied reaction steps, the fructosyl ring bound in the -1 position was observed to have a 4E conformation in the oxocarbonium ion-like transition state. Based on the SCC-DFTB/MM simulations of sucrose complexes of wildtype and D191A mutant FT, Asp191 is shown to be responsible for the productive sugar conformation (at subsite -1) required for catalysis. A key interaction, Asp119⋯nucleophile⋯1-OH (substrate), is proposed to facilitate the formation of fructosyl-enzyme intermediate. This is the first computational study for understanding the FOS synthesis process, and it can be applicable to related FOS-synthesizing enzymes.

Quantum Mechanics/Molecular Mechanics Simulations Identify the Ring-Opening Mechanism of Creatininase.

Creatininase catalyzes the conversion of creatinine (a biosensor for kidney function) to creatine via a two-step mechanism: water addition followed by ring opening. Water addition is common to other known cyclic amidohydrolases, but the precise mechanism for ring opening is still under debate. The proton donor in this step is either His178 or a water molecule bound to one of the metal ions, and the roles of His178 and Glu122 are unclear. Here, the two possible reaction pathways have been fully examined by means of combined quantum mechanics/molecular mechanics simulations at the SCC-DFTB/CHARMM22 level of theory. The results indicate that His178 is the main catalytic residue for the whole reaction and explain its role as proton shuttle during the ring-opening step. In the first step, His178 provides electrostatic stabilization to the gem-diolate tetrahedral intermediate. In the second step, His178 abstracts the hydroxyl proton of the intermediate and delivers it to the cyclic amide nitrogen, leading to ring opening. The latter is the rate-limiting step with a free energy barrier of 18.5 kcal/mol, in agreement with the experiment. We find that Glu122 must be protonated during the enzyme reaction, so that it can form a stable hydrogen bond with its neighboring water molecule. Simulations of the E122Q mutant showed that this replacement disrupts the H-bond network formed by three conserved residues (Glu34, Ser78, and Glu122) and water, increasing the energy barrier. Our computational studies provide a comprehensive explanation for previous structural and kinetic observations, including why the H178A mutation causes a complete loss of activity but the E122Q mutation does not.

Acetylcholinesterase inhibitory activity and molecular docking study of steroidal alkaloids from Holarrhena pubescens barks.

An alkaloidal extract of the bark of Holarrhena pubescens showed several inhibition zones of acetylcholinesterase (AChE) inhibitor, using a bioautographic assay. Activity-guided fractionation afforded three new steroidal alkaloids, mokluangins A-C (1-3), together with three known compounds, antidysentericine (4), holaphyllamine (5), methylholaphyllamine (6). All structures were elucidated by analysis of NMR and MS spectroscopic data. Compound 2 showed moderate antibacterial activity against Bacillus subtilis and Escherichia coli with the MIC value of 16 µg/mL, while compound 3 exhibited moderate selective activity against E. coli with the MIC value of 16 µg/mL. In addition, compounds 1-4 also showed strong AChE inhibiting activity with IC50 values ranging from 1.44 to 23.22 µM. Molecular docking calculations were also performed and the results demonstrated that all compounds can bind at the aromatic gorge of AChE with estimated binding free energies correlated well with the in vitro inhibitory profiles. Hydrophobic and hydrogen bonding interactions contribute mainly to the binding of the alkaloids where the substituents at C-3 serving as key functional groups for the AChE inhibition. Our results will allow the development of new AChE-inhibitors based on steroidal alkaloid skeleton bearing the cyclic amide moiety.

A DFT study of the unusual substrate-assisted mechanism of Serratia marcescens chitinase B reveals the role of solvent and mutational effect on catalysis.

Serratia marcescens chitinase B (SmChiB) catalyzes the hydrolysis of ß-1,4-glycosidic bond, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. In this paper, the catalytic mechanism of SmChiB has been investigated by using density functional theory. The details of two consecutive steps (glycosylation and deglycosylation), the structures and energetics along the whole catalytic reaction, and the roles of solvent molecules as well as some conserved SmChiB residues (Asp142, Tyr214, Asp215, and Arg294) during catalysis are highlighted. Our calculations show that the formation of the oxazolinium cation intermediate in the glycosylation step was found to be a rate-determining step (with a barrier of 23 kcal/mol), in line with our previous computational studies (Jitonnom et al., 2011, 2014). The solvent water molecules have a significant effect on a catalytic efficiency in the degycosylation step: the catalytic water is essentially placed in a perfect position for nucleophic attack by hydrogen bond network, lowering the barrier height of this step from 11.3 kcal/mol to 2.9 kcal/mol when more water molecules were introduced. Upon the in silico mutations of the four conserved residues, their mutational effects on the relative stability of the reaction intermediates and the computed energetics can be obtained by comparing with the wild-type results. Mutations of Tyr214 to Phe or Ala have shown a profound effect on the relative stability of the oxazolinium intermediate, emphasizing a direct role of this residue in destabilizing the intermediate. In line with the experiment that the D142A mutation leads to almost complete loss of SmChiB activity, this mutation greatly decreases the stability of the intermediate, resulting in a very large increase in the activation barrier up to 50 kcal/mol. The salt-bridges residues (Asp215 and Arg294) were also found to play a role in stabilizing the oxazolinium intermediate.

QM/MM free-energy simulations of reaction in Serratia marcescens Chitinase B reveal the protonation state of Asp142 and the critical role of Tyr214.

Serratia marcescens Chitinase B (ChiB), belonging to the glycosidase family 18 (GH18), catalyzes the hydrolysis of ß-1,4-glycosidic bond, with retention of configuration, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. Here, both elementary steps (glycosylation and deglycosylation) of the ChiB-catalyzed reaction are investigated by means of combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations at the SCC-DFTB/CHARMM22 level of theory. We examine the influence of the Asp142 protonation state on the reaction and the role that this residue performs in the reaction. Our simulations show that reaction with a neutral Asp142 is preferred and demonstrate that this residue provides electrostatic stabilization of the oxazolinium ion intermediate formed in the reaction. Insight into the conformational itinerary ((1,4)B↔(4)H5↔(4)C1) adopted by the substrate (bound in subsite -1) along the preferred reaction pathway is also provided by the simulations. The relative energies of the stationary points found along the reaction pathway calculated with SCC-DFTB and B3LYP were compared. The results suggest that SCC-DFTB is an accurate method for estimating the relative barriers for both steps of the reaction; however, it was found to overestimate the relative energy of an intermediate formed in the reaction when compared with the higher level of theory. Glycosylation is suggested to be a rate-determining step in the reaction with calculated overall reaction free-energy barrier of 20.5 kcal/mol, in a reasonable agreement with the 16.1 kcal/mol barrier derived from the experiment. The role of Tyr214 in catalysis was also investigated with the results, indicating that the residue plays a critical role in the deglycosylation step of the reaction. Simulations of the enzyme-product complex were also performed with an unbinding event suggested to have been observed, affording potential new mechanistic insight into the release of the product of ChiB.

Comparative study on activation mechanism of carboxypeptidase A1, A2 and B: first insights from steered molecular dynamics simulations.

Different forms of procarboxypeptidases (PCPs) zymogens are observed experimentally to show different rates and modes of activation: PCPA1 shows a slow, biphasic, activation pathway compared to PCPA2 and PCPB which have a faster, monotonic activation behavior. Detailed mechanisms involved in activating these zymogen forms to the active enzymes are not well understood yet. In this work, three PCP zymogens (subtypes A1, A2 and B) were in silico converted into the primary cleavage state of zymogens using available X-ray structures. Based on these cleaved forms of zymogen, we are able to investigate their spontaneous dissociation process of the prosegment from its associated enzyme domain using steered molecular dynamics simulation. The simulations revealed the highest rupture force in PCPB followed by PCPA2 and PCPA1. We also found that the cleavage substantially destabilizes most of the hydrogen bonds at the prosegment-enzyme interface in each zymogen structure. The mechanisms of the prosegment unbinding seem to be similar in both PCPA1 and PCPB but different in PCPA2: PCPA1 and PCPB show first rupture in the connecting segment followed by the globular domain, while PCPA2 conversely shows first rupture in the globular domain and then in the connecting segment. Our simulations have included the dynamic and long range conformational effects taking place after the first proteolytic cleavage in PCPs, providing first insights into the activation of carboxypeptidase A1, A2 and B.

Computational design of peptide inhibitor based on modifications of proregion from Plutella xylostella midgut trypsin.

Many proteases are produced as zymogens bearing the N-terminal proregions acting both as intramolecular chaperones and as protease inhibitors. The latter role of the proregions as potent and specific inhibitors of their associated protease has been demonstrated in various peptidases and therefore has been targeted for alternative pest control. Here, we isolated amino acid sequence of Plutella xylostella midgut trypsin from the larvae of diamondback moth and tested in silico for its inhibitory activity toward propeptide models using computational modeling and docking. The propeptide models (AAAPGHR, AAAPGRR, AAAPGKR, AAPGHRI, APGHRIV, PGHRIVG, AAAAPGH, and AAAAAPG) were designed based on histidine-mutated and frame-shifted modifications of the 7-amino-acid proregion (AAAPGHR) of the Plutella xylostella trypsin. Among the eight peptides, AAAPGRR was found to give the best docking scores, showing a strong binding to the cognate enzyme. In addition, the obtained structure of trypsin-AAAPGRR complex was found to share a similar binding mode with a crystal structure of plant protease inhibitor complex. Our results may guide the experiment for the design of future peptide inhibitor with specificity and selectivity for the target enzyme.

Quantum mechanics/molecular mechanics modeling of substrate-assisted catalysis in family 18 chitinases: conformational changes and the role of Asp142 in catalysis in ChiB.

Family 18 chitinases catalyze the hydrolysis of ß-1,4-glycosidic bonds in chitin. The mechanism has been proposed to involve the formation of an oxazolinium ion intermediate via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile (instead of an enzyme residue). Here, we have modeled the first step of the chitin hydrolysis catalyzed by Serratia marcescens chitinase B for the first time using a combined quantum mechanics/molecular mechanics approach. The calculated reaction barriers based on multiple snapshots are 15.8-19.8 kcal mol(-1) [B3LYP/6-31+G(d)//AM1-CHARMM22], in good agreement with the activation free energy of 16.1 kcal mol(-1) derived from experiment. The enzyme significantly stabilizes the oxazolinium intermediate. Two stable conformations ((4)C(1)-chair and B(3,O)-boat) of the oxazolinium ion intermediate in subsite -1 were unexpectedly observed. The transition state structure has significant oxacarbenium ion-like character. The glycosyl residue in subsite -1 was found to follow a complex conformational pathway during the reaction ((1,4)B → [(4)H(5)/(4)E](++) → (4)C(1) ↔ B(3,O)), indicating complex conformational behavior in glycoside hydrolases that utilize a substrate-assisted catalytic mechanism. The D142N mutant is found to follow the same wild-type-like mechanism: the calculated barriers for reaction in this mutant (16.0-21.1 kcal mol(-1)) are higher than in the wild type, in agreement with the experiment. Asp142 is found to be important in transition state and intermediate stabilization.

Influence of metal cofactors and water on the catalytic mechanism of creatininase-creatinine in aqueous solution from molecular dynamics simulation and quantum study.

The reaction mechanism of creatinine-creatininase binding to form creatine as a final product has been investigated by using a combined ab initio quantum mechanical/molecular mechanical approach and classical molecular dynamics (MD) simulations. In MD simulations, an X-ray crystal structure of the creatininase/creatinine was modified for creatininase/creatinine complexes and the MD simulations were run for free creatininase and creatinine in water. MD results reveal that two X-ray water molecules can be retained in the active site as catalytic water. The binding free energy from Molecular Mechanics Poisson-Boltzmann Surface Area calculation predicted the strong binding of creatinine with Zn2+, Asp45 and Glu183. Two step mechanisms via Mn2+/Zn2+ (as in X-ray structure) and Zn2+/Zn2+ were proposed for water adding step and ring opening step with two catalytic waters. The pathway using synchronous transit methods with local density approximations with PWC functional for the fragment in the active region were obtained. Preferable pathway Zn2+/Zn2+ was observed due to lower activation energy in water adding step. The calculated energy in the second step for both systems were comparable with the barrier of 26.03 and 24.44 kcal/mol for Mn2+/Zn2+ and Zn2+/Zn2+, respectively.