Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidase

AAO (aryl-alcohol oxidase) provides H?O? in fungal degradation of lignin, a process of high biotechnological interest. The crystal structure of AAO does not show open access to the active site, where different aromatic alcohols are oxidized. In the present study we investigated substrate diffusion and oxidation in AAO compared with the structurally related CHO (choline oxidase). Cavity finder and ligand diffusion simulations indicate the substrate-entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr?2. Mixed QM (quantum mechanics)/MM (molecular mechanics) studies combined with site-directed mutagenesis showed two active-site catalytic histidine residues, whose substitution strongly decreased both catalytic and transient-state reduction constants for p-anisyl alcohol in the H502A (over 1800-fold) and H546A (over 35-fold) variants. Combination of QM/MM energy profiles, protonation predictors, molecular dynamics, mutagenesis and pH profiles provide a robust answer regarding the nature of the catalytic base. The histidine residue in front of the FAD ring, AAO His??2 (and CHO His???), acts as a base. For the two substrates assayed, it was shown that proton transfer preceded hydride transfer, although both processes are highly coupled. No stable intermediate was observed in the energy profiles, in contrast with that observed for CHO. QM/MM, together with solvent KIE (kinetic isotope effect) results, suggest a non-synchronous concerted mechanism for alcohol oxidation by AAO.     

Structure of human biliverdin IXbeta reductase, an early fetal bilirubin IXbeta producing enzyme

Biliverdin IXbeta reductase (BVR-B) catalyzes the pyridine nucleotide-dependent production of bilirubin-IXbeta, the major heme catabolite during early fetal development. BVR-B displays a preference for biliverdin isomers without propionates straddling the C10 position, in contrast to biliverdin IXalpha reductase (BVR-A), the major form of BVR in adult human liver. In addition to its tetrapyrrole clearance role in the fetus, BVR-B has flavin and ferric reductase activities in the adult. We have solved the structure of human BVR-B in complex with NADP+ at 1.15 A resolution. Human BVR-B is a monomer displaying an alpha/beta dinucleotide binding fold. The structures of ternary complexes with mesobiliverdin IValpha, biliverdin IXalpha, FMN and lumichrome show that human BVR-B has a single substrate binding site, to which substrates and inhibitors bind primarily through hydrophobic interactions, explaining its broad specificity. The reducible atom of both biliverdin and flavin substrates lies above the reactive C4 of the cofactor, an appropriate position for direct hydride transfer. BVR-B discriminates against the biliverdin IXalpha isomer through steric hindrance at the bilatriene side chain binding pockets. The structure also explains the enzyme's preference for NADP(H) and its B-face stereospecificity.     

Role of active site histidines in the two half-reactions of the aryl-alcohol oxidase catalytic cycle

The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k(cat)/K(m(Al)) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O(2) constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH(-) to O(2), without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H(+) proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H(2)O(2). No solvent KIE was found for O(2) reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k(cat)/K(m(Ox)), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations.     

Catalytic mechanism of aldose reductase studied by the combined potentials of quantum mechanics and molecular mechanics

The catalytic reduction of

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-glyceraldehyde to glycerol by aldose reductase has been investigated with the combined potentials of quantum mechanics (QM) and molecular mechanics (MM) to resolve the question of whether Tyr48 or His110 serves as the proton donor during catalysis. Site directed mutagenesis studies favor Tyr48 as the proton donor while the presence of a water channel linking the Nδ1 of His110 to the bulk solvent suggests that His110 is the proton donor. Utilizing the combined potentials of QM and MM, the binding mode of substrate

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-glyceraldehyde was investigated by optimizing the local geometry of Asp43, Lys77, Tyr48, His110 and NADPH at the active site of aldose reductase. Reaction pathways for the reduction of

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-glyceraldehyde to glycerol were then constructed by treating both Tyr48 and His110 as proton donors. Comparison of energetics obtained from the reaction pathways suggests His110 to be the proton donor. Based on these findings, a reduction mechanism of

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-glyceraldehyde to glycerol is described.     

Determinants of reactivity and selectivity in soluble epoxide hydrolase from quantum mechanics/molecular mechanics modeling

Soluble epoxide hydrolase (sEH) is an enzyme involved in drug metabolism that catalyzes the hydrolysis of epoxides to form their corresponding diols. sEH has a broad substrate range and shows high regio- and enantioselectivity for nucleophilic ring opening by Asp333. Epoxide hydrolases therefore have potential synthetic applications. We have used combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations (at the AM1/CHARMM22 level) and high-level ab initio (SCS-MP2) QM/MM calculations to analyze the reactions, and determinants of selectivity, for two substrates: trans-stilbene oxide (t-SO) and trans-diphenylpropene oxide (t-DPPO). The calculated free energy barriers from the QM/MM (AM1/CHARMM22) umbrella sampling MD simulations show a lower barrier for phenyl attack in t-DPPO, compared with that for benzylic attack, in agreement with experiment. Activation barriers in agreement with experimental rate constants are obtained only with the highest level of QM theory (SCS-MP2) used. Our results show that the selectivity of the ring-opening reaction is influenced by several factors, including proximity to the nucleophile, electronic stabilization of the transition state, and hydrogen bonding to two active site tyrosine residues. The protonation state of His523 during nucleophilic attack has also been investigated, and our results show that the protonated form is most consistent with experimental findings. The work presented here illustrates how determinants of selectivity can be identified from QM/MM simulations. These insights may also provide useful information for the design of novel catalysts for use in the synthesis of enantiopure compounds.     

A theoretical investigation of the functional role of the axial methionine ligand of the Cu(A) site in cytochrome c oxidase

The functional roles of the amino acid residues of the Cu(A) site in bovine cytochrome c oxidase (CcO) were investigated by utilizing hybrid quantum mechanics (QM)/molecular mechanics (MM) calculations. The energy levels of the molecular orbitals (MOs) involving Cu d(zx) orbitals unexpectedly increased, as compared with those found previously with a simplified model system lacking the axial Met residue (i.e., Cu(2)S(2)N(2)). This elevation of MO energies stemmed from the formation of the anti-bonding orbitals, which are generated by hybridization between the d(zx) orbitals of Cu ions and the p-orbitals of the S and O atoms of the axial ligands. To clarify the roles of the axial Met ligand, the inner-sphere reorganization energies of the Cu(A) site were computed, with the Met residue assigned to either the QM or MM region. The reorganization energy slightly increased when the Met residue was excluded from the QM region. The existing experimental data and the present structural modeling study also suggested that the axial Met residue moderately increased the redox potential of the Cu(A) site. Thus, the role of the Met may be to regulate the electron transfer rate through the fine modulation of the electronic structure of the Cu(A) "platform", created by two Cys/His residues coordinated to the Cu ions. This regulation would provide the optimum redox potential/reorganization energy of the Cu(A) site, and thereby facilitate the subsequent cooperative reactions, such as the proton pump and the enzymatic activity, of CcO. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Proton transfer in carbonic anhydrase is controlled by electrostatics rather than the orientation of the acceptor

Combined quantum mechanical/molecular mechanical (QM/MM) simulations are carried out to analyze factors that dictate the proton transfer in carbonic anhydrase II (CAII), an enzyme that has been used as a prototypical example of long-range proton transfers in biomolecules. In contrast to the long-held conjecture in the experimental literature, the computed potentials of mean force (PMF) suggest that the proton transfer in CAII is not very sensitive to the orientation of the acceptor group (His 64) and, therefore, the number of water molecules that bridge the donor (zinc-water) and acceptor groups. Perturbative analysis indicates that a series of polar and charged residues close to the transfer pathways make the dominant contribution to the barrier and exothermicity of the proton transfer reaction, thus supporting the proposal from previous studies of Warshel and co-workers using a somewhat simpler QM/MM model that electrostatic interactions play a major role in the proton transfer in CAII. The PMF results are in striking contrast to previous analysis using the same QM/MM method but an ensemble of minimum energy path (MEP) calculations, which found a steep dependence of the barrier height on the number of bridging water molecules. Analysis of the configurations sampled in the PMF and MEP simulations suggests that this difference arises because the PMF simulations sample a largely stepwise mechanism while the local MEP calculations artificially favored concerted transfers due to the specific protocol used to generate the initial configurations. Therefore, this study presents a compelling argument for carrying out proper conformational sampling in the study of long-range proton transfers. Finally, we illustrate that Phi analysis, which has been widely used in protein folding studies, can potentially generate new mechanistic information for long-range proton transfers regarding the sequence of events. The results of the perturbation analysis and the Phi analysis provide opportunities for experimentally testing the mechanistic proposals from this study and our recent work in which a stepwise "proton hole" transfer pathway has been proposed.     

Hydrogen donor-acceptor fluctuations from kinetic isotope effects: a phenomenological model

Kinetic isotope effects (KIEs) and their temperature dependence can probe the structural and dynamic nature of enzyme-catalyzed proton or hydride transfers. The molecular interpretation of their temperature dependence requires expensive and specialized quantum mechanics/molecular mechanics (QM/MM) calculations to provide a quantitative molecular understanding. Currently available phenomenological models use a nonadiabatic assumption that is not appropriate for most hydride and proton-transfer reactions, while others require more parameters than the experimental data justify. Here we propose a phenomenological interpretation of KIEs based on a simple method to quantitatively link the size and temperature dependence of KIEs to a conformational distribution of the catalyzed reaction. This model assumes adiabatic hydrogen tunneling, and by fitting experimental KIE data, the model yields a population distribution for fluctuations of the distance between donor and acceptor atoms. Fits to data from a variety of proton and hydride transfers catalyzed by enzymes and their mutants, as well as nonenzymatic reactions, reveal that steeply temperature-dependent KIEs indicate the presence of at least two distinct conformational populations, each with different kinetic behaviors. We present the results of these calculations for several published cases and discuss how the predictions of the calculations might be experimentally tested. This analysis does not replace molecular QM/MM investigations, but it provides a fast and accessible way to quantitatively interpret KIEs in the context of a Marcus-like model.     

Reaction-path energetics and kinetics of the hydride transfer reaction catalyzed by dihydrofolate reductase

We have studied the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) and the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH); the substrate is 5-protonated 7,8-dihydrofolate, and the product is tetrahydrofolate. The potential energy surface is modeled by a combined quantum mechanical-molecular mechanical (QM/MM) method employing Austin model 1 (AM1) and a simple valence bond potential for 69 QM atoms and employing the CHARMM22 and TIP3P molecular mechanics force fields for the other 21 399 atoms; the QM and MM regions are joined by two boundary atoms treated by the generalized hybrid orbital (GHO) method. All simulations are carried out using periodic boundary conditions at neutral pH and 298 K. In stage 1, a reaction coordinate is defined as the difference between the breaking and forming bond distances to the hydride ion, and a quasithermodynamic free energy profile is calculated along this reaction coordinate. This calculation includes quantization effects on bound vibrations but not on the reaction coordinate, and it is used to locate the variational transition state that defines a transition state ensemble. Then, the key interactions at the reactant, variational transition state, and product are analyzed in terms of both bond distances and electrostatic energies. The results of both analyses support the conclusion derived from previous mutational studies that the M20 loop of DHFR makes an important contribution to the electrostatic stabilization of the hydride transfer transition state. Third, transmission coefficients (including recrossing factors and multidimensional tunneling) are calculated and averaged over the transition state ensemble. These averaged transmission coefficients, combined with the quasithermodynamic free energy profile determined in stage 1, allow us to calculate rate constants, phenomenological free energies of activation, and primary and secondary kinetic isotope effects. A primary kinetic isotope effect (KIE) of 2.8 has been obtained, in good agreement with the experimentally determined value of 3.0 and with the value 3.2 calculated previously. The primary KIE is mainly a consequence of the quantization of bound vibrations. In contrast, the secondary KIE, with a value of 1.13, is almost entirely due to dynamical effects on the reaction coordinate, especially tunneling.     

Low-barrier hydrogen bond hypothesis in the catalytic triad residue of serine proteases: correlation between structural rearrangement and chemical shifts in the acylation process

To elucidate the catalytic advantage of the low-barrier hydrogen bond (LBHB), we analyze the hydrogen bonding network of the catalytic triad (His57-Asp102-Ser195) of serine protease trypsin, one of the best examples of the LBHB reaction mechanism. Especially, we focus on the correlation between the change of the chemical shifts and the structural rearrangement of the active site in the acylation process. To clarify LBHB, we evaluate the two complementary properties. First, we calculate the NMR chemical shifts of the imidazole ring of His57 by the gauge-including atomic orbital (GIAO) approach within the ab initio QM/MM framework. Second, the free energy profile of the proton transfer from His57 to Asp102 in the tetrahedral intermediate is obtained by ab initio QM/MM calculations combined with molecular dynamics free energy perturbation (MD-FEP) simulations. The present analyses reveal that the calculated shifts reasonably reproduce the observed values for (1)H chemical shift of H(epsilon)(1) and H(delta)(1) in His57. The (15)N and (13)C chemical shifts are also consistent with the experiments. It is also shown that the proton between His57 and Asp102 is localized at the His57 side. This largely downfield chemical shift is originated from the strong electrostatic interaction, not a covalent-like bonding character between His57 and Asp102. Also, it is proved that a slight downfield character of H(epsilon)(1) is originated from a electrostatic interaction between His57 and the backbone carbonyl group of Val213 and Ser214. These downfield chemical shifts are observed only when the tetrahedral intermediate is formed in the acylation process.     

An Alternative Mechanism for the Methylation of Phosphoethanolamine Catalyzed by Plasmodium falciparum Phosphoethanolamine Methyltransferase

The phosphobase methylation pathway catalyzed by the phosphoethanolamine methyltransferase in Plasmodium falciparum (PfPMT), the malaria parasite, offers an attractive target for anti-parasitic drug development. PfPMT methylates phosphoethanolamine (pEA) to phosphocholine for use in membrane biogenesis. Quantum mechanics and molecular mechanics (QM/MM) calculations tested the proposed reaction mechanism for methylation of pEA involving the previously identified Tyr-19–His-132 dyad, which indicated an energetically unfavorable mechanism. Instead, the QM/MM calculations suggested an alternative mechanism involving Asp-128. The reaction coordinate involves the stepwise transfer of a proton to Asp-128 via a bridging water molecule followed by a typical S_n2-type methyl transfer from S-adenosylmethionine to pEA. Functional analysis of the D128A, D128E, D128Q, and D128N PfPMT mutants shows a loss of activity with pEA but not with the final substrate of the methylation pathway. X-ray crystal structures of the PfPMT-D128A mutant in complex with S-adenosylhomocysteine and either pEA or phosphocholine reveal how mutation of Asp-128 disrupts a hydrogen bond network in the active site. The combined QM/MM, biochemical, and structural studies identify a key role for Asp-128 in the initial step of the phosphobase methylation pathway in Plasmodium and provide molecular insight on the evolution of multiple activities in the active site of the PMT.     

Exploring the RING-Catalyzed Ubiquitin Transfer Mechanism by MD and QM/MM Calculations

Ubiquitylation is a universal mechanism for controlling cellular functions. A large family of ubiquitin E3 ligases (E3) mediates Ubiquitin (Ub) modification. To facilitate Ub transfer, RING E3 ligases bind both the substrate and ubiquitin E2 conjugating enzyme (E2) linked to Ub via a thioester bond to form a catalytic complex. The mechanism of Ub transfer catalyzed by RING E3 remains elusive. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations, we characterized this catalytic mechanism in detail. The three-dimensional model of dimeric RING E3 ligase RNF4 RING, E2 ligase UbcH5A, Ub and the substrate SUMO2 shows close contact between the substrate and Ub transfer catalytic center. Deprotonation of the substrate lysine by D117 on UbcH5A occurs with almost no energy barrier as calculated by MD and QM/MM calculations. Then, the side chain of the activated lysine gets close to the thioester bond via a conformation change. The Ub transfer pathway begins with a nucleophilic addition that forms an oxyanion intermediate of a 4.23 kcal/mol energy barrier followed by nucleophilic elimination, resulting in a Ub modified substrate by a 5.65 kcal/mol energy barrier. These results provide insight into the mechanism of RING-catalyzed Ub transfer guiding the discovery of Ub system inhibitors.     

Theoretical Insights into Catalytic Mechanism of Protein Arginine Methyltransferase 1

Protein arginine methyltransferase 1 (PRMT1), the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet) as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical S_N2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.     

QM/MM investigation of structure and spectroscopic properties of a vanadium-containing peroxidase

We present a comprehensive analysis of the most likely ground state configuration of the resting state of vanadium dependent chloroperoxidase (VCPO) based on quantum mechanics/molecular mechanics (QM/MM) evaluations of ground state properties, UV-vis spectra and NMR chemical shifts. Within the QM/MM framework, density functional theory (DFT) calculations are used to characterize the resting state of VCPO via time-dependent density functional theory (TD-DFT) calculations of electronic excitation energies and NMR chemical shifts. Comparison with available experimental data allows us to determine the most likely protonation state of VCPO, a state which results in a doubly protonated axial oxygen, a site largely stabilized by hydrogen bonds. We found that the bulk of the protein that is beyond the immediate layer surrounding the cofactor, has an important electrostatic effect on the absorption maximum. Through examination of frontier orbitals, we analyze the nature of two bound water molecules and the extent to which relevant residues in the active site influence the spectroscopy calculations.     

Second step of hydrolytic dehalogenation in haloalkane dehalogenase investigated by QM/MM methods

Mechanistic studies on the hydrolytic dehalogenation catalyzed by haloalkane dehalogenases are of importance for environmental and industrial applications. Here, Car-Parrinello (CP) and ONIOM hybrid quantum-mechanical/molecular mechanics (QM/MM) are used investigate the second reaction step of the catalytic cycle, which comprises a general base-catalyzed hydrolysis of an ester intermediate (EI) to alcohol and free enzyme. We focus on the enzyme LinB from Sphingomonas paucimobilis UT26, for which the X-ray structure at atomic resolution is available. In agreement with previous proposals, our calculations suggest that a histidine residue (His272), polarized by glutamate (Glu132), acts as a base, accepting a proton from the catalytic water molecule and transferring it to an alcoholate ion. The reaction proceeds through a metastable tetrahedral intermediate, which shows an easily reversed reaction to the EI. In the formation of the products, the protonated aspartic acid (Asp108) can easily adopt conformation of the relaxed state found in the free enzyme. The overall free energy barrier of the reaction calculated by potential of the mean force integration using CP-QM/MM calculations is equal to 19.5 +/- 2 kcal . mol(-1). The lowering of the energy barrier of catalyzed reaction with respect to the water reaction is caused by strong stabilization of the reaction intermediate and transition state and their preorganization by electrostatic field of the enzyme.     

Density functional theory calculations on entire proteins for free energies of binding: Application to a model polar binding site

In drug optimization calculations, the molecular mechanics Poisson‐Boltzmann surface area (MM‐PBSA) method can be used to compute free energies of binding of ligands to proteins. The method involves the evaluation of the energy of configurations in an implicit solvent model. One source of errors is the force field used, which can potentially lead to large errors due to the restrictions in accuracy imposed by its empirical nature. To assess the effect of the force field on the calculation of binding energies, in this article we use large‐scale density functional theory (DFT) calculations as an alternative method to evaluate the energies of the configurations in a “QM‐PBSA” approach. Our DFT calculations are performed with a near‐complete basis set and a minimal parameter implicit solvent model, within the self‐consistent calculation, using the ONETEP program on protein–ligand complexes containing more than 2600 atoms. We apply this approach to the T4‐lysozyme double mutant L99A/M102Q protein, which is a well‐studied model of a polar binding site, using a set of eight small aromatic ligands. We observe that there is very good correlation between the MM and QM binding energies in vacuum but less so in the solvent. The relative binding free energies from DFT are more accurate than the ones from the MM calculations, and give markedly better agreement with experiment for six of the eight ligands. Furthermore, in contrast to MM‐PBSA, QM‐PBSA is able to correctly predict a nonbinder. Proteins 2014; 82:3335–3346. © 2014 Wiley Periodicals, Inc.     

The reaction mechanism of paraoxon hydrolysis by phosphotriesterase from combined QM/MM simulations

Molecular dynamics simulations employing combined quantum mechanical and molecular mechanical (QM/MM) potentials have been carried out to investigate the reaction mechanism of the hydrolysis of paraoxon by phosphotriesterase (PTE). We used a dual-level QM/MM approach that synthesizes accurate results from high-level electronic structure calculations with computational efficiency of semiempirical QM/MM potentials for free energy simulations. In particular, the intrinsic (gas-phase) energies of the active site in the QM region are determined by using density functional theory (B3LYP) and second-order M?ller-Plesset perturbation theory (MP2) and the molecular dynamics free energy simulations are performed by using the mixed AM1:CHARMM potential. The simulation results suggest a revised mechanism for the phosphotriester hydrolysis mechanism by PTE. The reaction free energy profile is mirrored by structural motions of the binuclear metal center in the active site. The two zinc ions occupy a compact conformation with an average zinc-zinc distance of 3.5 +/- 0.1 A in the Michaelis complex, whereas it is elongated to 5.3 +/- 0.3 A at the transition state and product state. The substrate is loosely bound to the more exposed zinc ion (Znbeta2+) at an average distance of 3.8 A +/- 0.3 A. The P=O bond of the substrate paraoxon is activated by adopting a tight coordination to the Znbeta2+, releasing the coordinate to the bridging hydroxide ion and increasing its nucleophilicity. It was also found that a water molecule enters into the binding pocket of the loosely bound binuclear center, originally occupied by the nucleophilic hydroxide ion. We suggest that the proton of this water molecule is taken up by His254 at low pH or released to the solvent at high pH, resulting in a hydroxide ion that pulls the Znbeta2+ ion closer to form the compact configuration and restores the resting state of the enzyme.     

Catalytic mechanism and product specificity of rubisco large subunit methyltransferase: QM/MM and MD investigations

Molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations have been carried out in an investigation of Rubisco large subunit methyltransferase (LSMT). It was found that the appearance of a water channel is required for the stepwise methylation by S-adenosylmethionine (AdoMet). The water channel appears in the presence of AdoMet (LSMT.Lys-NH3+.AdoMet), but is not present immediately after methyl transfer (LSMT.Lys-N(Me)H2+.AdoHcy). The water channel allows proton dissociation from both LSMT.AdoMet.Lys-NH3+ and LSMT.AdoMet.Lys-N(Me)H2+. The water channel does not appear for proton dissociation from LSMT.AdoMet.Lys-N(Me)2H+, and a third methyl transfer does not occur. By QM/MM, the calculated free energy barrier of the first methyl transfer reaction catalyzed by LSMT (Lys-NH2 + AdoMet --> Lys-N(Me)H2+ + AdoHcy) is DeltaG++ = 22.8 +/- 3.3 kcal/mol. This DeltaG++ is in remarkable agreement with the value 23.0 kcal/mol calculated from the experimental rate constant (6.2 x 10-5 s-1). The calculated DeltaG++ of the second methyl transfer reaction (AdoMet + Lys-N(Me)H --> AdoHcy + Lys-N(Me)2H+) at the QM/MM level is 20.5 +/- 3.6 kcal/mol, which is in agreement with the value 22.0 kcal/mol calculated from the experimental rate constant (2.5 x 10-4 s-1). The third methyl transfer (Lys-N(Me)2 + AdoMet --> Lys-N(Me)3+ + AdoHcy) is associated with an allowed DeltaG++ of 25.9 +/- 3.2 kcal/mol. However, this reaction does not occur because a water channel does not form to allow the proton dissociation of Lys-N(Me)2H+. Future studies will determine whether the product specificity of lysine (mono, di, and tri) methyltransferases is determined by the formation of water channels.     

Binding free energy calculation with QM/MM hybrid methods for Abl-Kinase inhibitor

We report a Quantum mechanics/Molecular Mechanics–Poisson-Boltzmann/ Surface Area (QM/MM-PB/SA) method to calculate the binding free energy of c-Abl human tyrosine kinase by combining the QM and MM principles where the ligand is treated quantum mechanically and the rest of the receptor by classical molecular mechanics. To study the role of entropy and the flexibility of the protein ligand complex in a solvated environment, molecular dynamics calculations are performed using a hybrid QM/MM approach. This work shows that the results of the QM/MM approach are strongly correlated with the binding affinity. The QM/MM interaction energy in our reported study confirms the importance of electronic and polarization contributions, which are often neglected in classical MM-PB/SA calculations. Moreover, a comparison of semi-empirical methods like DFTB-SCC, PM3, MNDO, MNDO-PDDG, and PDDG-PM3 is also performed. The results of the study show that the implementation of a DFTB-SCC semi-empirical Hamiltonian that is derived from DFT gives better results than other methods. We have performed such studies using the AMBER molecular dynamic package for the first time. The calculated binding free energy is also in agreement with the experimentally determined binding affinity for c-Abl tyrosine kinase complex with Imatinib.     

Carbinolamine formation and dehydration in a DNA repair enzyme active site

In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1) (27.2 kJ mol(-1)) for the carbinolamine formation reaction and 44.5 kcal mol(-1) (186 kJ mol(-1)) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1) (62.8 kJ mol(-1)) in the forward (formation) reaction and 19 kcal mol(-1) (79.5 kJ mol(-1)) for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N-terminal amine.