The substrate-bound crystal structure of a Baeyer-Villiger monooxygenase exhibits a Criegee-like conformation

The Baeyer-Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP(+) and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP(+) and FAD, to 2.4 ? resolution. This structure shows a drastic rotation of the NADP(+) cofactor in comparison to previously reported NADP(+)-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP(+) permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme's chemospecificity, to a location above the flavin-peroxide adduct where catalysis occurs.     

Convergence in the QM‐only and QM/MM modeling of enzymatic reactions: A case study for acetylene hydratase

We report systematic quantum mechanics‐only (QM‐only) and QM/molecular mechanics (MM) calculations on an enzyme‐catalyzed reaction to assess the convergence behavior of QM‐only and QM/MM energies with respect to the size of the chosen QM region. The QM and MM parts are described by density functional theory (typically B3LYP/def2‐SVP) and the CHARMM force field, respectively. Extending our previous work on acetylene hydratase with QM regions up to 157 atoms (Liao and Thiel, J. Chem. Theory Comput. 2012, 8, 3793), we performed QM/MM geometry optimizations with a QM region M4 composed of 408 atoms, as well as further QM/MM single‐point calculations with even larger QM regions up to 657 atoms. A charge deletion analysis was conducted for the previously used QM/MM model ( M3a , with a QM region of 157 atoms) to identify all MM residues with strong electrostatic contributions to the reaction energetics (typically more than 2 kcal/mol), which were then included in M4 . QM/MM calculations with this large QM region M4 lead to the same overall mechanism as the previous QM/MM calculations with M3a , but there are some variations in the relative energies of the stationary points, with a mean absolute deviation (MAD) of 2.7 kcal/mol. The energies of the two relevant transition states are close to each other at all levels applied (typically within 2 kcal/mol), with the first (second) one being rate‐limiting in the QM/MM calculations with M3a ( M4 ). QM‐only gas‐phase calculations give a very similar energy profile for QM region M4 (MAD of 1.7 kcal/mol), contrary to the situation for M3a where we had previously found significant discrepancies between the QM‐only and QM/MM results (MAD of 7.9 kcal/mol). Extension of the QM region beyond M4 up to M7 (657 atoms) leads to only rather small variations in the relative energies from single‐point QM‐only and QM/MM calculations (MAD typically about 1–2 kcal/mol). In the case of acetylene hydratase, a model with 408 QM atoms thus seems sufficient to achieve convergence in the computed relative energies to within 1–2 kcal/mol.Copyright © 2013 Wiley Periodicals, Inc.     

Comparison of linear-scaling semiempirical methods and combined quantum mechanical/molecular mechanical methods for enzymic reactions. II. An energy decomposition analysis

QM/MM methods have been developed as a computationally feasible solution to QM simulation of chemical processes, such as enzyme-catalyzed reactions, within a more approximate MM representation of the condensed-phase environment. However, there has been no independent method for checking the quality of this representation, especially for highly nonisotropic protein environments such as those surrounding enzyme active sites. Hence, the validity of QM/MM methods is largely untested. Here we use the possibility of performing all-QM calculations at the semiempirical PM3 level with a linear-scaling method (MOZYME) to assess the performance of a QM/MM method (PM3/AMBER94 force field). Using two model pathways for the hydride-ion transfer reaction of the enzyme dihydrofolate reductase studied previously (Titmuss et al., Chem Phys Lett 2000, 320, 169-176), we have analyzed the reaction energy contributions (QM, QM/MM, and MM) from the QM/MM results and compared them with analogous-region components calculated via an energy partitioning scheme implemented into MOZYME. This analysis further divided the MOZYME components into Coulomb, resonance and exchange energy terms. For the model in which the MM coordinates are kept fixed during the reaction, we find that the MOZYME and QM/MM total energy profiles agree very well, but that there are significant differences in the energy components. Most significantly there is a large change (approximately 16 kcal/mol) in the MOZYME MM component due to polarization of the MM region surrounding the active site, and which arises mostly from MM atoms close to (<10 A) the active-site QM region, which is not modelled explicitly by our QM/MM method. However, for the model where the MM coordinates are allowed to vary during the reaction, we find large differences in the MOZYME and QM/MM total energy profiles, with a discrepancy of 52 kcal/mol between the relative reaction (product-reactant) energies. This is largely due to a difference in the MM energies of 58 kcal/mol, of which we can attribute approximately 40 kcal/mol to geometry effects in the MM region and the remainder, as before, to MM region polarization. Contrary to the fixed-geometry model, there is no correlation of the MM energy changes with distance from the QM region, nor are they contributed by only a few residues. Overall, the results suggest that merely extending the size of the QM region in the QM/MM calculation is not a universal solution to the MOZYME- and QM/MM-method differences. They also suggest that attaching physical significance to MOZYME Coulomb, resonance and exchange components is problematic. Although we conclude that it would be possible to reparameterize the QM/MM force field to reproduce MOZYME energies, a better way to account for both the effects of the protein environment and known deficiencies in semiempirical methods would be to parameterize the force field based on data from DFT or ab initio QM linear-scaling calculations. Such a force field could be used efficiently in MD simulations to calculate free energies.     

Interfacing Q-Chem and CHARMM to perform QM/MM reaction path calculations

A hybrid quantum mechanical/molecular mechanical (QM/MM) potential energy function with Hartree-Fock, density functional theory (DFT), and post-HF (RIMP2, MP2, CCSD) capability has been implemented in the CHARMM and Q-Chem software packages. In addition, we have modified CHARMM and Q-Chem to take advantage of the newly introduced replica path and the nudged elastic band methods, which are powerful techniques for studying reaction pathways in a highly parallel (i.e., parallel/parallel) fashion, with each pathway point being distributed to a different node of a large cluster. To test our implementation, a series of systems were studied and comparisons were made to both full QM calculations and previous QM/MM studies and experiments. For instance, the differences between HF, DFT, MP2, and CCSD QM/MM calculations of H2O...H2O, H2O...Na+, and H2O...Cl- complexes have been explored. Furthermore, the recently implemented polarizable Drude water model was used to make comparisons to the popular TIP3P and TIP4P water models for doing QM/MM calculations. We have also computed the energetic profile of the chorismate mutase catalyzed Claisen rearrangement at various QM/MM levels of theory and have compared the results with previous studies. Our best estimate for the activation energy is 8.20 kcal/mol and for the reaction energy is -23.1 kcal/mol, both calculated at the MP2/6-31+G(d)//MP2/6-31+G(d)/C22 level of theory.     

Mechanism of C-terminal intein cleavage in protein splicing from QM/MM molecular dynamics simulations

Protein splicing is a post-translational process in which a biologically inactive protein is activated by the release of a segment denoted as an intein. The process involves four steps. In the third, the scission of the intein takes place after the cyclization of the last amino acid of the segment, an asparagine. Little is known about the chemical reaction necessary for this cyclization. Experiments demonstrate that two histidines (the penultimate amino acid of the intein, and a histidine located 10 amino acids upstream) are relevant in the cyclization of the asparagine. We have investigated the mechanism and determinants of reaction in the GyrA intein focusing on the requirements for asparagine activation for its cyclization. First, the influence that the protonation states of these two histidines have on the orientation of the asparagine side chain is investigated by means of molecular dynamics simulation. Molecular dynamics simulations using the CHARMM27 force field were carried out on the three possible protonation states for each of these two histidines. The results indicate that the only protonation state in which the conformation of the system is suitable for cyclization is when the penultimate histidine is fully protonated (positively charged), and the upstream histidine is in the His(ε) neutral tautomeric form. The free energy profile for the reaction in which the asparagine is activated by a proton transfer to the upstream histidine is presented, computed by hybrid quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics at the SCCDFTB/CHARMM27 level of theory. The calculated free energy barrier for the reaction is 19.0 kcal mol(-1). B3LYP/6-31+G(d) QM/MM single-point calculations give a qualitatively a similar energy profile, although with somewhat higher energy barriers, in good agreement with the value derived from experiment of 25 kcal mol(-1) at 60 °C. QM/MM molecular dynamics simulations of the reactant, activated reactant and intermediate states highlight the importance of the Arg181-Val182-Asp183 segment in catalysing the reaction. Overall, the results indicate that nucleophilic activation of the asparagine for its cyclization by the upstream histidine acting as the base is a plausible mechanism for the C-terminal cleavage in protein splicing.     

Peptide hydrolysis catalyzed by matrix metalloproteinase 2: a computational study

The MMP-2 reaction mechanism is investigated by using different computational methodologies. First, quantum mechanical (QM) calculations are carried out on a cluster model of the active site bound to an Ace-Gly approximately Ile-Nme peptide. Along the QM reaction path, a Zn-bound water molecule attacks the Gly carbonyl group to give a tetrahedral intermediate. The breaking of the C-N bond is completed thanks to the Glu 404 residue that shuttles a proton from the water molecule to Ile-N atom. The gas-phase QM energy barrier is quite low ( approximately 14 kcal/mol), thus suggesting that the essential catalytic machinery is included in the cluster model. A similar reaction path occurs in the MMP-2 catalytic domain bound to an octapeptide substrate according to hybrid QM and molecular mechanical (QM/MM) geometry optimizations. However, the rupture of the Gly( P 1) approximately Ile( P 1') amide bond is destabilized in the static QM/MM calculations, owing to the positioning of the Ile( P 1') side chain inside the MMP-2 S 1' pocket and to the inability of simple energy miminization methodologies to properly relax complex systems. Molecular dynamics simulations show that these steric limitations are overcome easily through structural fluctuations. The energetic effect of structural fluctuations is taken into account by combining QM energies with average MM Poisson-Boltzmann free energies, resulting in a total free energy barrier of 14.8 kcal/mol in good agreement with experimental data. The rate-determining event in the MMP-2 mechanism corresponds to a H-bond rearrangement involving the Glu 404 residue and/or the Glu 404-COOH --> N-Ile( P 1') proton transfer. Overall, the present computational results and previous experimental data complement each other well in order to provide a detailed view of the MMPs catalytic mechanism.     

Antibiotic deactivation by a dizinc beta-lactamase: mechanistic insights from QM/MM and DFT studies

Hybrid quantum mechanical/molecular mechanical (QM/MM) methods and density functional theory (DFT) were used to investigate the initial ring-opening step in the hydrolysis of moxalactam catalyzed by the dizinc L1 beta-lactamase from Stenotrophomonas maltophilia. Anchored at the enzyme active site via direct metal binding as suggested by a recent X-ray structure of an enzyme-product complex (Spencer, J.; et al. J. Am. Chem. Soc. 2005, 127, 14439), the substrate is well aligned with the nucleophilic hydroxide that bridges the two zinc ions. Both QM/MM and DFT results indicate that the addition of the hydroxide nucleophile to the carbonyl carbon in the substrate lactam ring leads to a metastable intermediate via a dominant nucleophilic addition barrier. The potential of mean force obtained by SCC-DFTB/MM simulations and corrected by DFT/MM calculations yields a reaction free energy barrier of 23.5 kcal/mol, in reasonable agreement with the experimental value of 18.5 kcal/mol derived from kcat of 0.15 s(-1). It is further shown that zinc-bound Asp120 plays an important role in aligning the nucleophile, but accepts the hydroxide proton only after the nucleophilic addition. The two zinc ions are found to participate intimately in the catalysis, consistent with the proposed mechanism. In particular, the Zn(1) ion is likely to serve as an "oxyanion hole" in stabilizing the carbonyl oxygen, while the Zn(2) ion acts as an electrophilic catalyst to stabilize the anionic nitrogen leaving group.     

Molecular dynamics and free energy perturbation study of hydride-ion transfer step in dihydrofolate reductase using combined quantum and molecular mechanical model

We used molecular dynamics simulation and free energy perturbation (FEP) methods to investigate the hydride-ion transfer step in the mechanism for the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of a novel substrate by the enzyme dihydrofolate reductase (DHFR). The system is represented by a coupled quantum mechanical and molecular mechanical (QM/MM) model based on the AM1 semiempirical molecular orbital method for the reacting substrate and NADPH cofactor fragments, the AMBER force field for DHFR, and the TIP3P model for solvent water. The FEP calculations were performed for a number of choices for the QM system. The substrate, 8-methylpterin, was treated quantum mechanically in all the calculations, while the larger cofactor molecule was partitioned into various QM and MM regions with the addition of “link” atoms (F, CH₃, and H). Calculations were also carried out with the entire NADPH molecule treated by QM. The free energies of reaction and the net charges on the NADPH fragments were used to determine the most appropriate QM/MM model. The hydride-ion transfer was also carried out over several FEP pathways, and the QM and QM/MM component free energies thus calculated were found to be state functions (i.e., independent of pathway). A ca. 10 kcal/mol increase in free energy for the hydride-ion transfer with an activation barrier of ca. 30 kcal/mol was calculated. The increase in free energy on the hydride-ion transfer arose largely from the QM/MM component. Analysis of the QM/MM energy components suggests that, although a number of charged residues may contribute to the free energy change through long-range electrostatic interactions, the only interaction that can account for the 10 kcal/mol increase in free energy is the hydrogen bond between the carboxylate side chain of Glu30 (avian DHFR) and the activated (protonated) substrate. © 1998 John Wiley & Sons, Inc. J Comput Chem 19: 977–988, 1998     

Variational calculation of quantum mechanical/molecular mechanical free energy with electronic polarization of solvent

Quantum mechanical/molecular mechanical (QM/MM) free energy calculation presents a significant challenge due to an excessive number of QM calculations. A useful approach for reducing the computational cost is that based on the mean field approximation to the QM subsystem. Here, we describe such a mean-field QM/MM theory for electronically polarizable systems by starting from the Hartree product ansatz for the total system and invoking a variational principle of free energy. The MM part is then recast to a classical polarizable model by introducing the charge response kernel. Numerical test shows that the potential of mean force (PMF) thus obtained agrees quantitatively with that obtained from a direct QM/MM calculation, indicating the utility of self-consistent mean-field approximation. Next, we apply the obtained method to prototypical reactions in several qualitatively different solvents and make a systematic comparison of polarization effects. The results show that in aqueous solution the PMF does not depend very much on the water models employed, while in nonaqueous solutions the PMF is significantly affected by explicit polarization. For example, the free energy barrier for a phosphoryl dissociation reaction in acetone and cyclohexane is found to increase by more than 10 kcal/mol when switching the solvent model from an empirical to explicitly polarizable one. The reason for this is discussed based on the parametrization of empirical nonpolarizable models.     

QM/MM study of the taxadiene synthase mechanism

Combined quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the reaction mechanism of taxadiene synthase (TXS). TXS catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to taxadiene (T) and four minor cyclic products. All these products originate from the deprotonation of carbocation intermediates. The reaction profiles for the conversion of GGPP to T as well as to minor products were calculated for different configurations of relevant TXS carbocation complexes. The QM region was treated at the M06-2X/TZVP level, while the CHARMM27 force field was used to describe the MM region. The QM/MM calculations suggest a reaction pathway for the conversion of GGPP to T, which slightly differs from previous proposals regarding the number of reaction steps and the conformation of the carbocations. The QM/MM results also indicate that the formation of minor products via water-assisted deprotonation of the carbocations is highly exothermic, by about −7 to −23 kcal/mol. Curiously, however, the computed barriers and reaction energies indicate that the formation of some of the minor products is more facile than the formation of T. Thus, the present QM/MM calculations provide detailed insights into possible reaction pathways and into the origin of the promiscuity of TXS, but they do not reproduce the product distribution observed experimentally. © 2019 Wiley Periodicals, Inc.     

A novel quantum mechanical/molecular mechanical approach to the free energy calculation for isomerization of glycine in aqueous solution

The free energy change associated with the isomerization reaction of glycine in water solution has been studied by a hybrid quantum mechanical/molecular mechanical (QM/MM) approach combined with the theory of energy representation (QM/MM-ER) recently developed. The solvation free energies for both neutral and zwitterionic form of glycine have been determined by means of the QM/MM-ER simulation. The contributions of the electronic polarization and the fluctuation of the QM solute to the solvation free energy have been investigated. It has been found that the contribution of the density fluctuation of the zwitterionic solute is estimated as -4.2 kcal/mol in the total solvation free energy of -46.1 kcal/mol, while that of the neutral form is computed as -3.0 kcal/mol in the solvation free energy of -15.6 kcal/mol. The resultant free energy change associated with the isomerization of glycine in water has been obtained as -7.8 kcal/mol, in excellent agreement with the experimental data of -7.3 or -7.7 kcal/mol, implying the accuracy of the QM/MM-ER approach. The results have also been compared with those computed by other methodologies such as the polarizable continuum model and the classical molecular simulation. The efficiency and advantage of the QM/MM-ER method has been discussed.     

Catalytic mechanism and product specificity of the histone lysine methyltransferase SET7/9: an ab initio QM/MM-FE study with multiple initial structures

Histone lysine methylation is emerging as an important mechanism to regulate chromatin structure and gene activity. To provide theoretical understanding of its reaction mechanism and product specificity, ab initio quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations and molecular dynamics simulations have been carried out to investigate the histone lysine methyltransferase SET7/9. It is found that the methyl-transfer reaction catalyzed by SET7/9 is a typical in-line S(N)2 nucleophilic substitution reaction with a transition state of 70% dissociative character. The calculated average free energy barrier at the MP2(6-31+G) QM/MM level is 20.4 +/- 1.1 kcal/mol, consistent with the activation barrier of 20.9 kcal/mol estimated from the experimental reaction rate. The barrier fluctuation has a strong correlation with the nucleophilic attack distance and angle in the reactant complex. The calculation results show that the product specificity of SET7/9 as a monomethyltransferase is achieved by disrupting the formation of near-attack conformations for the dimethylation reaction.     

Mechanism of proteolysis in matrix metalloproteinase‐2 revealed by QM/MM modeling

The mechanism of enzymatic peptide hydrolysis in matrix metalloproteinase‐2 (MMP‐2) was studied at atomic resolution through quantum mechanics/molecular mechanics (QM/MM) simulations. An all‐atom three‐dimensional molecular model was constructed on the basis of a crystal structure from the Protein Data Bank (ID: 1QIB), and the oligopeptide Ace‐Gln‐Gly～Ile‐Ala‐Gly‐Nme was considered as the substrate. Two QM/MM software packages and several computational protocols were employed to calculate QM/MM energy profiles for a four‐step mechanism involving an initial nucleophilic attack followed by hydrogen bond rearrangement, proton transfer, and C? N bond cleavage. These QM/MM calculations consistently yield rather low overall barriers for the chemical steps, in the range of 5–10 kcal/mol, for diverse QM treatments (PBE0, B3LYP, and BB1K density functionals as well as local coupled cluster treatments) and two MM force fields (CHARMM and AMBER). It, thus, seems likely that product release is the rate‐limiting step in MMP‐2 catalysis. This is supported by an exploration of various release channels through QM/MM reaction path calculations and steered molecular dynamics simulations. © 2015 Wiley Periodicals, Inc.     

Why urea eliminates ammonia rather than hydrolyzes in aqueous solution

A joint QM/MM and ab initio study on the decomposition of urea in the gas phase and in aqueous solution is reported. Numerous possible mechanisms of intramolecular decomposition and hydrolysis have been explored; intramolecular NH3 elimination assisted by a water molecule is found to have the lowest activation energy. The solvent effects were elucidated using the TIP4P explicit water model with free energy perturbation calculations in conjunction with QM/MM Monte Carlo simulations. The explicit representation of the solvent was found to be essential for detailed resolution of the mechanism, identification of the rate-determining step, and evaluation of the barrier. The assisting water molecule acts as a hydrogen shuttle for the first step of the elimination reaction. The forming zwitterionic intermediate, H3NCONH, participates in 8-9 hydrogen bonds with water molecules. Its decomposition is found to be the rate-limiting step, and the overall free energy of activation for the decomposition of urea in water is computed to be approximately 37 kcal/mol; the barrier for hydrolysis by an addition/elimination mechanism is found to be approximately 40 kcal/mol. The differences in the electronic structure of the transition states of the NH3 elimination and hydrolysis were examined via natural bond order analysis. Destruction of urea's resonance stabilization during hydrolysis via an addition/elimination mechanism and its preservation in the rearrangement to the H3NCONH intermediate were identified as important factors in determining the preferred reaction route.     

A QM/MM simulation study of transamination reaction at the active site of aspartate aminotransferase: Free energy landscape and proton transfer pathways

Transaminase is a key enzyme for amino acid metabolism, which reversibly catalyzes the transamination reaction with the help of PLP (pyridoxal 5^' -phosphate) as its cofactor. Here we have investigated the mechanism and free energy landscape of the transamination reaction involving the aspartate transaminase (AspTase) enzyme and aspartate-PLP (Asp-PLP) complex using QM/MM simulation and metadynamics methods. The reaction is found to follow a stepwise mechanism where the active site residue Lys258 acts as a base to shuttle a proton from α -carbon (CA) to imine carbon (C4A) of the PLP-Asp Schiff base. In the first step, the Lys258 abstracts the CA proton of the substrate leading to the formation of a carbanionic intermediate which is followed by the reprotonation of the Asp-PLP Schiff base at C4A atom by Lys258. It is found that the free energy barrier for the proton abstraction by Lys258 and that for the reprotonation are 17.85 and 3.57 kcal/mol, respectively. The carbanionic intermediate is 7.14 kcal/mol higher in energy than the reactant. Hence, the first step acts as the rate limiting step. The present calculations also show that the Lys258 residue undergoes a conformational change after the first step of transamination reaction and becomes proximal to C4A atom of the Asp-PLP Schiff base to favor the second step. The active site residues Tyr70* and Gly38 anchor the Lys258 in proper position and orientation during the first step of the reaction and stabilize the positive charge over Lys258 generated at the intermediate step.     

Theoretical perspectives on the reaction mechanism of serine proteases: the reaction free energy profiles of the acylation process

The reaction mechanism of serine proteases (trypsin), which catalyze peptide hydrolysis, is studied theoretically by ab initio QM/MM electronic structure calculations combined with Molecular Dynamics-Free Energy Perturbation calculations. We have calculated the entire reaction free energy profiles of the first reaction step of this enzyme (acylation process). The present calculations show that the rate-determining step of the acylation is the formation of the tetrahedral intermediate, and the breakdown of this intermediate has a small energy barrier. The calculated activation free energy for the acylation is approximately 17.8 kcal/mol at QM/MM MP2/(aug)-cc-pVDZ//HF/6-31(+)G/AMBER level, and this reaction is an exothermic process. MD simulations of the enzyme-substrate (ES) complex and the free enzyme in aqueous phase show that the substrate binding induces slight conformational changes around the active site, which favor the alignment of the reactive fragments (His57, Asp102, and Ser195) together in a reactive orientation. It is also shown that the proton transfer from Ser195 to His57 and the nucleophilic attack of Ser195 to the carbonyl carbon of the scissile bond of the substrate occur in a concerted manner. In this reaction, protein environment plays a crucial role to lowering the activation free energy by stabilizing the tetrahedral intermediate compared to the ES complex. The polarization energy calculations show that the enzyme active site is in a very polar environment because of the polar main chain contributions of protein. Also, the ground-state destabilization effect (steric strain) is not a major catalytic factor. The most important catalytic factor of stabilizing the tetrahedral intermediate is the electrostatic interaction between the active site and particular regions of protein: the main chain NH groups in Gly193 and Ser195 (so-called oxyanion hole region) stabilize negative charge generated on the carbonyl oxygen of the scissile bond, and the main chain carbonyl groups in Ile212 approximately Ser214 stabilize a positive charge generated on the imidazole ring of His57.     

Systematic QM/MM investigation of factors that affect the cytochrome P450-catalyzed hydrogen abstraction of camphor

The hydrogen abstraction reaction of camphor in cytochrome P450(cam) has been investigated in the native enzyme environment by combined quantum mechanical/molecular mechanical (QM/MM) calculations and in the gas phase by density functional calculations. This work has been motivated by contradictory published QM/MM results. In an attempt to pinpoint the origin of these discrepancies, we have systematically studied the factors that may affect the computed barriers, including the QM/MM setup, the optimization procedures, and the choice of QM region, basis set, and protonation states. It is found that the ChemShell and QSite programs used in the published QM/MM calculations yield similar results at given geometries, and that the discrepancies mainly arise from two technical issues (optimization protocols and initial system preparation) that need to be well controlled in QM/MM work. In the course of these systematic investigations, new mechanistic insights have been gained. The crystallographic water 903 placed near the oxo atom of Compound I lowers the hydrogen abstraction barrier by ca. 4 kcal/mol, and thus acts as a catalyst for this reaction. Spin density may appear at the A-propionate side chain of the heme if the carboxylate group is not properly screened, which might be expected to happen during protein dynamics, but not in static equilibrium situations. There is no clear correlation between the computed A-propionate spin density and the hydrogen abstraction barrier, and hence, no support for a previously proposed side-chain mediated transition state stabilization mechanism. Standard QM/MM optimizations yield an A-propionate environment close to the X-ray structure only for protonated Asp297, and not for deprotonated Asp297, but the computed barriers are similar in both cases. An X-ray like A-propionate environment can also be obtained when deprotonated Asp297 is included in the QM region and His355 is singly protonated, but this Compound II-type species with a closed-shell porphyrin ring has a higher hydrogen abstraction barrier and should thus not be mechanistically relevant.     

Hybrid quantum mechanics/molecular mechanics-based molecular dynamics simulation of acid-catalyzed dehydration of polyols in liquid water

We use the conversion of protonated glycerol to acrolein for a case study of the mechanism of acid-catalyzed dehydration of polyols in aqueous environments. We employ hybrid Quamtum Mechanics/Molecular Mechanics Molecular Dynamics (QM/MM MD) simulations with biased sampling and perform free energy calculations for the elementary steps of the reaction. We investigate the effects of solvent dynamics and in particular the role of quantum mechanical water in the dehydration mechanism. We present results supporting a mechanism that proceeds via water-mediated proton transfers and thus through an enol intermediate. We find that the first dehydration may take place by two, low-energy pathways requiring, respectively, 20.9 and 18.8 kcal/mol of activation free energy. The second dehydration requires 19.9 kcal/mol of activation free energy while for the overall reaction we compute a free energy change of -8 kcal/mol.