A QM/MM study of the racemization of vinylglycolate catalyzed by mandelate racemase enzyme.

The experimentally postulated mechanism for the interconversion between (S)-vinylglycolate and (R)-vinylglycolate catalyzed by mandelate racemase enzyme consists of a two-step quite symmetric process through a dianionic enolic intermediate that is formed after the abstraction of the alpha-proton of vinylglycolate by a basic enzymatic residue and is then reprotonated by another residue. The challenging problem behind this reaction is how the enzyme manages to stabilize such an intermediate, that is, how it lowers enough the high pK(a) of the alpha-proton for the reaction to take place. The QM/MM simulations performed in this paper indicate that catalysis is based on the stabilization of the negative charge developed on the substrate along the reaction. We have identified three different reaction mechanisms starting from different quasi-degenerate structures of the substrate-enzyme complex. In two of them the stabilizing role is done by means of a catalytic proton transfer that avoids the formation of a dianionic intermediate, and they involve six steps instead of the two experimentally proposed. On the contrary, the third mechanism passes through a dianionic species stabilized by the concerted approach of a protonated enzymatic residue during the proton abstraction. The potential energy barriers theoretically found along these mechanisms are qualitatively in good agreement with the experimental free energy barriers determined for racemization of vinylglycolate and mandelate. The theoretical study of the effect of the mutation of Glu317 by Gln317 in the kinetics of the reaction reveals the important role in the catalysis of the hydrogen bond formed by Glu317 in the native enzyme, as only one of the mechanisms, the slower one, is able to produce the racemization in the active site of the mutant. However, we have found that this hydrogen bond is not an LBHB within our model.     

Ab initio explorative survey of the mechanism catalyzed by mandelate racemase

There is chemical and kinetic evidence that the mechanism of action of mandelate racemase occurs via two acid/base catalysts, one to abstract a proton from the -carbon and the second to deliver it back to the opposite face of the chiral center. Since the mechanistic details are not known, a few hypotheses have been put forward. Therefore we examine the viability of both concerted and sequential mechanisms on proton abstraction from the -carbon of mandelate, either without taking into account environmental effects or with the addition to the model system of several functional groups of the residues belonging to the active site, in order to restrain the partners to particular positions without actually imposing arbitrary constraints. The importance of the environment in screening the most highly charged groups in the substrate and in facilitating the -carbon-bound proton abstraction is evident. From the perusal of the geometries of the system along the reaction energy profile, it seems that there is no tendency toward a concerted mechanism: the proton delivery from mandelate to Lys occurs and subsequently the extra proton on protonated histidine is in turn delivered to mandelate.     

Alanine racemase free energy profiles from global analyses of progress curves

Free energy profiles for alanine racemase from Bacillus stearothermophilus have been determined at pH 6.9 and 8.9 from global analysis of racemization progress curves. This required a careful statistical design due to the problems in finding the global minimum in mean square for a system with eight adjustable parameters (i.e., the eight rate constants that describe the stepwise chemical mechanism). The free energy profiles obtained through these procedures are supported by independent experimental evidence: (1). steady-state kinetic constants, (2). solvent viscosity dependence, (3). spectral analysis of reaction intermediates, (4). equilibrium overshoots for progress curves measured in D(2)O, and (5). the magnitudes of calculated intrinsic kinetic isotope effects. The free energy profiles for the enzyme are compared to those of the uncatalyzed and the PLP catalyzed reactions. At pH 6.9, PLP lowers the free energy of activation for deprotonation by 8.4 kcal/mol, while the inclusion of apoenzyme along with PLP additionally lowers it by 11 kcal/mol.     

Non-centrosymmetric coordination polymer with a highly hindered octahedral copper center bridged by mandelate

A novel chiral coordination polymer, [Cu(C(6)H(5)CH(OH)COO)(μ-C(6)H(5)CH(OH)COO)] (1-L and 1-D), was synthesized through a reaction of copper acetate with L-mandelic acid at room temperature. Although previously reported copper mandelate prepared by hydrothermal reaction was a centrosymmetric coordination polymer because of the racemization of mandelic acid, the current coordination polymer shows noncentrosymmetry and a completely different structure from that previously reported. The X-ray crystallography for 1-L revealed that the copper center of the compound showed a highly distorted octahedral structure bridged by a chiral mandelate ligand in the unusual coordination mode to construct a one-dimensional (1D) zigzag chain structure. These 1D chains interdigitated each other to give a layered structure as a result of the formation of multiple aromatic interactions and hydrogen bonds between hydroxyl and carboxylate moieties at mandelate ligands. The coordination polymer 1-L belongs to the noncentrosymmetric space group of C2 to show piezoelectric properties and second harmonic generation (SHG) activity.     

Ab initio quantum mechanical/molecular mechanical molecular dynamics simulation of enzyme catalysis: the case of histone lysine methyltransferase SET7/9

To elucidate enzyme catalysis through computer simulation, a prerequisite is to reliably compute free energy barriers for both enzyme and solution reactions. By employing on-the-fly Born-Oppenheimer molecular dynamics simulations with the ab initio quantum mechanical/molecular mechanical approach and the umbrella sampling method, we have determined free energy profiles for the methyl-transfer reaction catalyzed by the histone lysine methyltransferase SET7/9 and its corresponding uncatalyzed reaction in aqueous solution, respectively. Our calculated activation free energy barrier for the enzyme catalyzed reaction is 22.5 kcal/mol, which agrees very well with the experimental value of 20.9 kcal/mol. The difference in potential of mean force between a corresponding prereaction state and the transition state for the solution reaction is computed to be 30.9 kcal/mol. Thus, our simulations indicate that the enzyme SET7/9 plays an essential catalytic role in significantly lowering the barrier for the methyl-transfer reaction step. For the reaction in solution, it is found that the hydrogen bond network near the reaction center undergoes a significant change, and there is a strong shift in electrostatic field from the prereaction state to the transition state, whereas for the enzyme reaction, such an effect is much smaller and the enzyme SET7/9 is found to provide a preorganized electrostatic environment to facilitate the methyl-transfer reaction. Meanwhile, we find that the transition state in the enzyme reaction is a little more dissociative than that in solution.     

Modeling of the adsorption behavior and the chromatographic band profiles of enantiomers : Behavior of methyl mandelate on immobilized cellulose

The adsorption isotherms of (−)- and (+)-methyl mandelate from a hexane-isopropanol (90:10) solution were measured on a chromatographic column packed with 4-methylcellulose tribenzoate coated on silica. These isotherms are accounted for by a bi-Langmuir isotherm model, the two Langmuir terms having widely different initial slopes and saturation capacities, but each term having the same saturation capacity for the two enantiomers. The competitive isotherms were also measured. They are in excellent agreement with the prediction of a competitive bi-Langmuir model based on the single-component isotherms. The individual band profiles are in agreement with the profiles calculated from these isotherms. Thus, a simplified competitive isotherm can be used to model a separation on a chiral stationary phase the recognition mechanism of which is not well identified and the adsorption behavior of which is certainly not ideal.     

QM/MM study on catalytic mechanism of aspartate racemase from <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis> OT3

The enzyme aspartate racemase from Pyrococcus horikoshii OT3 catalyzes the interconversion between l- and d-Asp. In this work, we employed the hybrid QM/MM approach with the self-consistent charge-density functional tight binding (SCC-DFTB) model to study the catalytic mechanism for the conversion of l-Asp into d-Asp. The molecular dynamics simulation showed that the substrate l-Asp forms an extensive network of interactions with the active-site residues of the aspartate racemase through its side chain carboxylate, ammonium group, and α-carboxylate. The potential of mean force calculations confirmed that the racemization reaction involves two proton transfers (from the α-carbon to Cys194 and from Cys82 to the α-carbon), which occurs in a concerted way, although highly asynchronous. The calculated free energy of activation is 17.5 kcal/mol, which is consistent with the reaction rate measured from experiment. An electrostatic interaction analysis was performed to estimate the key role played by individual residues in stabilizing the transition state. The docking study on the binding of l-Asp and d-Asp to aspartate racemase indicates that this enzyme employs a “two-base” mechanism not a “one-base” mechanism.     

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.     

Enantioselective fluorescent recognition of mandelate by substituted BINOL in aqueous solutions

The fluorescent photo-induced electron transfer chemosensors (R)-1, (R)-2, (S)-1 and (S)-2 based on the 3,3′-positions of 1,1′-bi-2-naphthol were designed for their recognition of mandelate. The binding properties for mandelate were examined by the fluorescence spectra. The high fluorescence sensitivity and enantioselectivity make compound (R)-2 a practically useful sensor for the recognition of mandelate in CH₃OH/H₂O system (1:1, 0.01 M Tris–HCl buffer, pH 7.4).     

Transition state stabilization and alpha-amino carbon acidity in alanine racemase

Combined QM/MM simulations have been carried out to investigate the origin of the carbon acidity enhancement in the alanine racemization reaction catalyzed by alanine racemase (AlaR). The present studies show that enhancement of carbon acidity of alpha-amino acids by cofactor pyridoxal 5'-phosphate, PLP, with an unusual, unprotonated pyridine is due to solvation effects, in contrast to the intrinsic electron-withdrawing stabilization by the pyridinium ion to form a quinonoid intermediate. Alanine racemase further lowers the alpha-proton acidity and provides an overall 14-17 kcal/mol transition state stabilization. Our computational results are consistent with the hypothesis that the use of the unusual form of PLP cofactor in AlaR is to raise the free energy of the intermediate, thereby increasing the reprotonation rate and enhancing the enzyme selectivity for racemization.     

String method in collective variables: minimum free energy paths and isocommittor surfaces

A computational technique is proposed which combines the string method with a sampling technique to determine minimum free energy paths. The technique only requires to compute the mean force and another conditional expectation locally along the string, and therefore can be applied even if the number of collective variables kept in the free energy calculation is large. This is in contrast with other free energy sampling techniques which aim at mapping the full free energy landscape and whose cost increases exponentially with the number of collective variables kept in the free energy. Provided that the number of collective variables is large enough, the new technique captures the mechanism of transition in that it allows to determine the committor function for the reaction and, in particular, the transition state region. The new technique is illustrated on the example of alanine dipeptide, in which we compute the minimum free energy path for the isomerization transition using either two or four dihedral angles as collective variables. It is shown that the mechanism of transition can be captured using the four dihedral angles, but it cannot be captured using only two of them.     

Synthesis and enantioselective recognition of an (S)-BINOL-based colorimetric chemosensor for mandelate anions

New chiral receptors 1 and 2 based on (S)-BINOL and thiourea units were synthesized. The chiral recognition of receptors for chiral anions were studied by fluorescence, UV–vis, and ¹H NMR spectra. The results of the non-linear curve fitting indicated that the receptors and guest anions formed a 1:1 stoichiometric complex. The obvious color change of receptor 2 can be observed by the naked eye when the enantiomers of mandelate anions were added, which demonstrates that receptor 2 may be used as a colorimetric sensor for mandelate anions.     

Reaction mechanism of monoethanolamine with CO₂ in aqueous solution from molecular modeling

We present a theoretical study of the reaction mechanism of monoethanolamine (MEA) with CO? in an aqueous solution. We have used molecular orbital reaction pathway calculations to compute reaction free energy landscapes for the reaction steps involved in the formation of carbamic acids and carbamates. We have used the conductor-like polarizable continuum model to calculate reactant, product, and transition state geometries and vibrational frequencies within density functional theory (DFT). We have also computed single point energies for all stationary structures using a coupled cluster approach with singles, doubles, and perturbational triple excitations using the DFT geometries. Our calculations indicate that a two-step reaction mechanism that proceeds via a zwitterion intermediate to form carbamate is the most favorable reaction channel. The first step, leading to formation of the zwitterion, is found to be rate-determining, and the activation free energies are 12.0 (10.2) and 11.3 (9.6) kcal/mol using Pauling (Bondi) radii within the CPCM model at the CCSD(T)/6-311++G(d,p) and CCSD(T)/6-311++G(2df,2p) levels of theory, respectively, using geometries and vibrational frequencies obtained at the B3LYP/6-311++G(d,p) level of theory. These results are in reasonable agreement with the experimental value of about 12 kcal/mol. The second step is an acid-base reaction between a zwitterion and MEA. We have developed a microkinetic model to estimate the effective reaction order at intermediate concentrations. Our model predicts an equilibrium concentration for the zwitterion on the order of 10?11 mol/L, which explains why the existence of the zwitterion intermediate has never been detected experimentally. The effective reaction order from our model is close to unity, also in agreement with experiments. Complementary ab initio QM/MM molecular dynamics simulations with umbrella sampling have been carried out to determine the free energy profiles of zwitterion formation and proton transfer in solution; the results confirm that the formation of the zwitterion is rate-determining.     

A recipe for the computation of the free energy barrier and the lowest free energy path of concerted reactions

The recently introduced hills method (Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12562) is a powerful tool to compute the multidimensional free energy surface of intrinsically concerted reactions. We have extended this method by focusing our attention on localizing the lowest free energy path that connects the stable reactant and product states. This path represents the most probable reaction mechanism, similar to the zero temperature intrinsic reaction coordinate, but also includes finite temperature effects. The transformation of the multidimensional problem to a one-dimensional reaction coordinate allows for accurate convergence of the free energy profile along the lowest free energy path using standard free energy methods. Here we apply the hills method, our lowest free energy path search algorithm, and umbrella sampling to the prototype S(N)2 reaction. The hills method replaces the in many cases difficult problem of finding a good reaction coordinate with choosing relatively simple collective variables, such as the bond lengths of the broken and formed chemical bonds. The second part of the paper presents a guide to using the hills method, in which we test and fine-tune the method for optimal accuracy and efficiency using the umbrella sampling results as a reference.     

Atropoisomeric quinolinium salt promoting the access to both enantiomeric forms of methyl mandelate: a versatile NADH mimic

Asymmetric reduction of methyl benzoylformate by a new NADH mimic is reported; depending on the hydride source used to reduce the NAD+ precursor, NADH mimics so obtained lead to an inversion of enantioselectivity, affording either (R)-methyl mandelate in 88% ee or (S)-methyl mandelate in 78% ee.     

A comparison of methods to compute the potential of mean force.

Most processes occurring in a system are determined by the relative free energy between two or more states because the free energy is a measure of the probability of finding the system in a given state. When the two states of interest are connected by a pathway, usually called reaction coordinate, along which the free-energy profile is determined, this profile or potential of mean force (PMF) will also yield the relative free energy of the two states. Twelve different methods to compute a PMF are reviewed and compared, with regard to their precision, for a system consisting of a pair of methane molecules in aqueous solution. We analyze all combinations of the type of sampling (unbiased, umbrella-biased or constraint-biased), how to compute free energies (from density of states or force averaging) and the type of coordinate system (internal or Cartesian) used for the PMF degree of freedom. The method of choice is constraint-bias simulation combined with force averaging for either an internal or a Cartesian PMF degree of freedom.     

The catalytic activity of proline racemase: a quantum mechanical/molecular mechanical study

The enzyme proline racemase from the eukaryotic parasite Trypanosoma cruzi (responsible for endemic Chagas disease) catalyzes the reversible stereoinversion of chiral Calpha in proline. We employed a new combined quantum mechanical and molecular mechanical (QM/MM) potential to study the reaction mechanism of the enzyme. Three critical points were found: two almost isoenergetic minima (M1a and M2a), in which the enzyme is bound to L- and D-Pro, respectively, and a transition state (TSCa), unveiling a highly asynchronous concerted process. A systematic analysis was performed on the optimized geometries to point out the key role played by some residues in stabilizing the transition state.     

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.     

A Simple Model for Barrier Frequencies for Enzymatic Reactions

We present a simple model to rationalize the effects of environment on the reaction barrier frequencies derived from free energy profiles. These frequencies are relevant in deviations of a rate constant from its transition state theory value and in determining which environmental dynamics participate in the reaction. In particular, this simple model can be used to understand the changes in the reaction barrier frequencies of an enzymatic catalyzed reaction and the corresponding uncatalyzed process in aqueous solution, a change which has implications for dynamical environmental effects on the enzymatic reaction. Two possible cases are analyzed, in which the polarity (charge separation/localization) of the reacting system increases or decreases as the reaction advances. A simple modeling of the environment′s effects allows the explanation of an unusual “inverse” effect on the reaction frequencies, that is, a free energy barrier lowering accompanied by an increase of the reaction frequency, a behavior observed in some enzymes. The model predictions are successfully compared with results from full simulations for four different enzyme reactions.