Do dynamic effects play a significant role in enzymatic catalysis? A theoretical analysis of formate dehydrogenase.

A theoretical study of the protein dynamic effects on the hydride transfer between the formate anion and nicotinamide adenine dinucleotide (NAD(+)), catalyzed by formate dehydrogenase (FDH), is presented in this paper. The analysis of free downhill molecular dynamic trajectories, performed in the enzyme and compared with the reaction in aqueous solution, has allowed the study of the dynamic coupling between the reacting fragments and the protein or the solvent water molecules, as well as an estimation of the dynamic effect contribution to the catalytic effect from calculation of the transmission coefficient in the enzyme and in solution. The obtained transmission coefficients for the enzyme and in solution were 0.46±0.04 and 0.20±0.03, respectively. These values represent a contribution to catalysis of 0.5 kcal mol(-1), which, although small, is not negligible keeping in mind the low efficiency of FDH. The analysis of the reactive trajectories also reveals how the relative movements of some amino acids, mainly His332 and Arg284, precede and promote the chemical reaction. In spite of these movements, the time-dependent evolution of the electric field created by the enzyme on the key atoms of the reaction reveals a permanent field, which reduces the work required to reach the transition state, with a concomitant polarization of the cofactor. Finally, application of Grote-Hynes theory has allowed the identification of the modes responsible for the substrate-environment coupling, showing how some protein motions take place simultaneously with the reaction. Thus, the equilibrium approach would provide, in this case, an overestimation of the catalyzed rate constant.

Theoretical modeling of the reaction mechanism of phosphate monoester hydrolysis in alkaline phosphatase.

The reaction mechanism of phosphate monoester hydrolysis in alkaline phosphatase is analyzed by means of hybrid QM/MM simulations. A recently developed semiempirical Hamiltonian, AM1/d-PhoT, which takes into account the d orbitals on the phosphorus atom, has been employed. The reaction mechanism obtained is either associative or dissociative, depending on the size of the QM subsystem. The results are rationalized on the basis of the degree of charge transfer from the reacting fragments to the two zinc ions present in the active site, which has been observed to be dependent on whether or not metal atoms and their coordination spheres are included in the QM region. The description obtained using the largest QM region agrees with the picture obtained from experimental data.

Coupling of the guanosine glycosidic bond conformation and the ribonucleotide cleavage reaction: implications for barnase catalysis.

To examine the possible relationship of guanine-dependent GpA conformations with ribonucleotide cleavage, two potential of mean force (PMF) calculations were performed in aqueous solution. In the first calculation, the guanosine glycosidic (Gchi) angle was used as the reaction coordinate, and computations were performed on two GpA ionic species: protonated (neutral) or deprotonated (negatively charged) guanosine ribose O2 '. Similar energetic profiles featuring two minima corresponding to the anti and syn Gchi regions were obtained for both ionic forms. For both forms the anti conformation was more stable than the syn, and barriers of approximately 4 kcal/mol were obtained for the anti --> syn transition. Structural analysis showed a remarkable sensitivity of the phosphate moiety to the conformation of the Gchi angle, suggesting a possible connection between this conformation and the mechanism of ribonucleotide cleavage. This hypothesis was confirmed by the second PMF calculations, for which the O2 '--P distance for the deprotonated GpA was used as reaction coordinate. The computations were performed from two selected starting points: the anti and syn minima determined in the first PMF study of the deprotonated guanosine ribose O2'. The simulations revealed that the O2 ' attack along the syn Gchi was more favorable than that along the anti Gchi: energetically, significantly lower barriers were obtained in the syn than in the anti conformation for the O--P bond formation; structurally, a lesser O2 '--P initial distance, and a better suited orientation for an in-line attack was observed in the syn relative to the anti conformation. These results are consistent with the catalytically competent conformation of barnase-ribonucleotide complex, which requires a guanine syn conformation of the substrate to enable abstraction of the ribose H2 ' proton by the general base Glu73, thereby suggesting a coupling between the reactive substrate conformation and enzyme structure and mechanism.

Dynamic effects on reaction rates in a Michael addition catalyzed by chalcone isomerase. Beyond the frozen environment approach.

We present a detailed microscopic study of the dynamics of the Michael addition reaction leading from 6'-deoxychalcone to the corresponding flavanone. The reaction dynamics are analyzed for both the uncatalyzed reaction in aqueous solution and the reaction catalyzed by Chalcone Isomerase. By means of rare event simulations of trajectories started at the transition state, we have computed the transmission coefficients, obtaining 0.76 +/- 0.04 and 0.87 +/- 0.03, in water and in the enzyme, respectively. According to these simulations, the Michael addition can be seen as a formation of a new intramolecular carbon-oxygen bond accompanied by a charge transfer essentially taking place from the nucleophilic oxygen to the carbon atom adjacent to the carbonyl group (C (alpha)). As for intermolecular interactions, we find a very significant difference in the evolving solvation pattern of the nucleophilic oxygen in water and in the enzyme. While in the former medium this atom suffers an important desolvation, the enzyme provides, through variations in the distances with some residues and water molecules, an essentially constant electric field on this atom along the reaction progress. Grote-Hynes (GH) theory provides a useful framework to systematically analyze all the couplings between the reaction coordinate and the remaining degrees of freedom. This theory provides transmission coefficients in excellent agreement with the Molecular Dynamics estimations. In contrast, neither the frozen environment approach nor Kramers theory gives results of similar quality, especially in the latter case, where the transmission coefficients are severely underestimated. The (unusual) failure of the frozen environment approach signals the importance of some dynamical motions. Within the context of GH theory, analysis of the friction spectrum obtained in the enzymatic environment, together with normal-mode analysis, is used to identify those motions, of both the substrate and the environment, strongly coupled to the reaction coordinate and to classify them as dynamically active or inactive.

Using Grote-Hynes theory to quantify dynamical effects on the reaction rate of enzymatic processes. The case of methyltransferases.

Dynamical effects have recently received much attention in the context of the theoretical investigation of enzymatic catalysis. In this paper we use a combination of Grote-Hynes theory with quantum mechanical/molecular mechanical modeling that is a powerful tool to understand and quantify these dynamical effects in a particular enzyme, the glycine N-methyltransferase (GNMT). Comparison of the results obtained for this enzyme with another methyltransferase (catechol O-methyltransferase, COMT) allows us to understand the different nature of the coupling of the environment to the reaction coordinate as a function of the electrostatic interaction established by the reactive subsystem. The transmission coefficients obtained using Grote-Hynes theory are in excellent agreement with molecular dynamics estimations and show that the coupling is higher in GNMT than in COMT. The larger friction observed in GNMT is explained on the basis of the interaction established by the substrate in the active site. The larger value of the friction leads to a smaller value of the reaction frequency and thus also to a larger disagreement with the estimation of the transmission coefficient based on the frozen environment approach.

Reproductive success of Whiskered Tern Chlidonias hybrida in eastern Spain in relation to water level variation.

BACKGROUND: A study on the Whiskered Tern Chlidonias hybrida was carried out between 2002 and 2009 in wetlands of eastern Spain to evaluate how water level fluctuation affects its reproductive success (hatching, fledgling and breeding success). This species is catalogued as Vulnerable in Spain and has an unfavorable conservation status in Europe. METHODS: Our study includes 18 sampling areas from five wetlands, covering a total of 663 nests, 1,618 eggs, 777 nestlings and 225 fledglings. The colonies were visited at least twice per week in breeding period. The number of eggs and/or nestlings present in each nest were annotated each time the colonies were visited with the aim to compare the evolution of these parameters with time. Hatching success was calculated as the proportion of egg that hatched successfully. Fledgling success and breeding success were calculated as the proportion of chicks that fledged successfully and the proportion of eggs that produced fledglings. We used the Kruskal-Wallis test to analyze the differences in the dependent variables hatching, fledgling and breeding success among the wetlands and the sampling areas. We explored the relationship between the different reproductive success with the average fluctuation rate and the anchoring depth of nests, using statistics of the linear regression. RESULTS: It was observed that the reproductive success varied significantly in the interaction among the different categories of water level fluctuation and the different areas (using the Kruskal-Wallis test). Our records showed that pronounced variations in water level destroyed several nests, which affected the Whiskered Tern reproductive success. Considering all events that occurred in 18 areas, the mean (±SD) of nests, eggs and nestlings that were lost after water level fluctuations were of 25.60 ± 21.79%, 32.06 ± 27.58% and 31.91 ± 21.28% respectively, also including the effects of rain and predation. DISCUSSION: Unfavorable climatic events, such as strong wind, rain or hail, also caused the loss of nests, eggs and nestlings, even when wetland water levels remained constant. The influence of the anchorage depth of the nest and the water level fluctuation rate were analyzed and did not provide statistically significant results. It was not possible to establish a clear pattern on these latter variables, so further studies are needed to obtain more significant results. We propose to undertake similar studies in wetlands where the water level can be regulated, with the range of nest anchorage depth on the emergent vegetation being between 30 and 60 cm, which could improve the reproductive success in this kind of habitats. As recommendation, in water level controlled wetlands (that use sluices), it should not vary more than ±6 cm in a short time (1-2 days) once the nests are established since it negatively affects their reproductive success.

On Transition Structures for Hydride Transfer Step in Enzyme Catalysis. A Comparative Study on Models of Glutathione Reductase Derived from Semiempirical, HF, and DFT Methods.

As a model of the chemical reactions that take place in the active site of gluthatione reductase, the nature of the molecular mechanism for the hydride transfer step has been characterized by means of accurate quantum chemical characterizations of transition structures. The calculations have been carried out with analytical gradients at AM1 and PM3 semiempirical procedures, ab initio at HF level with 3-21G, 4-31G, 6-31G, and 6-31G basis sets and BP86 and BLYP as density functional methods. The results of this study suggest that the endo relative orientation on the substrate imposed by the active site is optimal in polarizing the C4-Ht bond and situating the system in the neighborhood of the quadratic region of the transition structure associated to the hydride transfer step on potential energy surface. The endo arrangement of the transition structure results in optimal frontier HOMO orbital interaction between NADH and FAD partners. The geometries of the transition structures and the corresponding transition vectors, that contain the fundamental information relating reactive fluctuation patterns, are model independent and weakly dependent on the level of theory used to determine them. A comparison between simple and complex molecular models shows that there is a minimal set of coordinates describing the essentials of hydride transfer step. The analysis of transition vector components suggests that the primary and secondary kinetic isotope effects can be strongly coupled, and this prompted the calculation of deuterium and tritium primary, secondary, and primary and secondary kinetic isotope effects. The results obtained agree well with experimental data and demonstrate this coupling.

Intrinsically Competitive Photoinduced Polycyclization and Double-Bond Shift through a Boatlike Conical Intersection.

A mechanistic view of the deactivation of photoexcited cycloocta-1,3,5,7-tetraene (COT*) through a novel type of conical intersection is provided by ab initio studies. As a consequence of this deactivation, the formation of semibullvalene (SBV) and of the double-bond-shifted isomer of COT are intrinsically bound. Both gas-phase and solution-phase experimental data are explained.

Theoretical exploration of the oxidative properties of a [(tren Me1)CuO2]+ adduct relevant to copper monooxygenase enzymes: insights into competitive dehydrogenation versus hydroxylation reaction pathways.

Singlet and triplet H-transfer reaction paths from C-H and N-H bonds were examined by means of DFT and spin-flip TD-DFT computations on the [(tren Me1)CuO2]+ adduct. The singlet energy surfaces allow its evolution towards H2O2 and an imine species. Whereas N-H cleavage appears to be a radical process, C-H rupture results in a carbocation intermediate stabilized by an adjacent N atom and an electrostatic interaction with the [CuIOOH] metal core. Upon injection of an additional electron, the latter species straightforwardly forms a hydroxylated product. Based on these computational results, a new mechanistic description of the reactivity of copper monooxygenases is proposed.

Coupling between protein and reaction dynamics in enzymatic processes: application of Grote-Hynes Theory to catechol O-methyltransferase.

The generalized Langevin equation (GLE)-based Grote-Hynes (GH) theory is used to calculate the transmission coefficients, kappa, for the methyl transfer from S-adenosylmethionine to catecholate both in aqueous solution and in the catechol O-methyltransferase active site. Values of kappa, which measures the deviation of the rate constants from the Transition State Theory (TST) predictions, are obtained by means of rare event molecular dynamics simulations. The results are 0.62 +/- 0.04 and 0.83 +/- 0.03 for the aqueous and enzymatic environments, respectively, while the Grote-Hynes predictions are 0.58 +/- 0.09 and 0.89 +/- 0.03, respectively. The Kramers theory estimates are much smaller, about 0.01 and 0.1, respectively. Thus, the enzymatic transmission coefficient is closer to TST predictions than the value obtained in solution. In addition, our results show that the enzymatic coefficient is also closer to its nonadiabatic (or frozen environment) limit than is the solution coefficient. These findings can be understood considering that, during the passage over the barrier top, there is a smaller coupling between the reactive system and the environment in the enzyme than in solution, as well as a smaller reorganization suffered by the enzyme. Analysis of the transition state friction kernel leads to the identification of some key vibrational modes governing the coupling between the two different environments and the reacting solute in the transition state region and insights on their relevance for the reaction dynamics' influence on the transmission coefficient.

Activation free energy of catechol O-methyltransferase. Corrections to the potential of mean force.

We use quantum mechanics/molecular mechanics (QM/MM) calculations to estimate the activation free energy for the chemical reaction catalyzed by catechol O-methyltransferase. While in many cases the activation free energy of a chemical process is directly determined by the potential of mean force associated with a particular reaction coordinate, here we have included several corrections that have been proposed in the literature. These include the free energy change associated with release of the reaction coordinate motion in the reactant state, consideration of the curvilinear nature of the reaction coordinate, and correction due to the classical treatment of molecular vibrations. In addition, since potentials of mean force are usually obtained from low levels of QM theory to describe the quantum subsystem, we have included an interpolated correction term to improve this description at small additional cost. This last correction term has a dramatic effect, improving the agreement between the theoretical predictions and the experimental value, while the other terms considered make only small contributions to this particular reaction.

Catalysis in glycine N-methyltransferase: testing the electrostatic stabilization and compression hypothesis.

Glycine N-methyltransferase (GNMT) is an S-adenosyl-l-methionine dependent enzyme that catalyzes glycine transformation to sarcosine. Here, we present a hybrid quantum mechanics/molecular mechanics (QM/MM) computational study of the reaction compared to the counterpart process in water. The process takes place through an SN2 mechanism in both media with a transition state in which the transferring methyl group is placed in between the donor (SAM) and the acceptor (the amine group of glycine). Comparative analysis of structural, electrostatic, and electronic characteristics of the in-solution and enzymatic transition states allows us to get a deeper insight into the origins of the enzyme's catalytic power. We found that the enzyme is able to stabilize the substrate in its more active basic form by means of a positively charged residue (Arg175) placed in the active site. However, the maximum stabilization is attained for the transition state. In this case, the enzyme is able to form stronger hydrogen bonds with the positively charged amine group. Finally, we show that in agreement with previous computational studies on other methyltransferases, there is no computational evidence for the compression hypothesis, as was formulated by Schowen (Hegazi, M. F., Borchardt, R. T., and Schowen, R. L. (1979) J. Am. Chem. Soc. 101, 4359-4365).

A theoretical analysis of rate constants and kinetic isotope effects corresponding to different reactant valleys in lactate dehydrogenase.

In some enzymatic systems large conformational changes are coupled to the chemical step, in such a way that dispersion of rate constants can be observed in single-molecule experiments, each corresponding to the reaction from a different reactant valley. Under this perspective here we present a computational study of pyruvate to lactate transformation catalyzed by lactate dehydrogenase. The reaction consists of a hydride transfer and a proton transfer that seem to take place concertedly. The degree of asynchronicity and the energy barrier depend on the particular starting reactant valley. In order to estimate rate constants we used a free energy perturbation technique adapted to follow the intrinsic reaction coordinate for several possible reaction paths. Tunneling effects are also obtained with a slightly modified version of the ensemble-averaged variational transition state theory with multidimensional tunneling contributions. According to our results the closure of the active site by means of a flexible loop can lead to the formation of different reactant complexes displaying different features in the disposition of some key residues (such as Arg109), interactions with the substrate and number of water molecules in the active site. The chemical step of the reaction takes place with a different reaction rate from each of these complexes. Finally, primary kinetic isotope effects for replacement of the transferring hydrogen of the cofactor with a deuteride are in good agreement with experimental observations, thus validating our methodology.

Improving the QM/MM Description of Chemical Processes: A Dual Level Strategy To Explore the Potential Energy Surface in Very Large Systems.

Potential energy surfaces are fundamental tools for the analysis of reaction mechanisms. The accuracy of these surfaces for reactions in very large systems is often limited by the size of the system even if hybrid quantum mechanics/molecular mechanics (QM/MM) strategies are employed. The large number of degrees of freedom of the system requires hundreds or even thousands of optimization steps to reach convergence. Reactions in condensed media (such as enzymes or solutions) are thus usually restricted to be analyzed using low level quantum mechanical methods, thus introducing a source of error in the description of the QM region. In this paper, an alternative method is proposed, coupled to the use of a micro/macroiteration algorithm during the optimization. In these algorithms, the number of microsteps involved in the QM region optimization is usually much smaller than the number of macrosteps required to optimize the MM region. Thus, we define a new potential energy surface in which the gas-phase energy of the QM subsystem and the interaction energy with the MM subsystem are calculated at different computational levels. The high computational level is restricted to the gas-phase energy, which is only requested during the microsteps. The dual level strategy is tested for two reactions in solution (the Menshutkin and the oxy-Cope reactions) and an enzymatic one (the nucleophilic substitution of 1,2-dichloroethane in DhlA). The performance of the proposed computational scheme seems to be quite promising for future applications in other systems.

A QM/MM Exploration of the Potential Energy Surface of Pyruvate to Lactate Transformation Catalyzed by LDH. Improving the Accuracy of Semiempirical Descriptions.

We present a QM/MM study of the potential energy surface of the pyruvate to lactate transformation catalyzed by L-lactate dehydrogenase. The transformation involves a hydride and a proton transfer which are followed by means of the corresponding antisymmetric combination of the distances from the hydrogen atom to the donor and the acceptor atoms. To discriminate among the possible reaction mechanisms we have considered different improvements of the AM1/MM description: reoptimization of the van der Waals parameters and inclusion of corrections to the QM energy associated with both transfer coordinates. The QM subsystem has been also enlarged to include charge-transfer effects from the substrate to some specific residues. In our best treatment, the transformation is described as a concerted process through a single transition structure in which the hydride transfer is more advanced than the proton transfer. From the methodological point of view, the correction schemes tested here improve the quality of the semiempirical potential energy surface although they also present deficiencies attributed to consideration of the proton and hydride transfer corrections as separable ones.

A comparative study of claisen and cope rearrangements catalyzed by chorismate mutase. An insight into enzymatic efficiency: transition state stabilization or substrate preorganization?

In this work we present a detailed analysis of the activation free energies and averaged interactions for the Claisen and Cope rearrangements of chorismate and carbachorismate catalyzed by Bacillus subtilischorismate mutase (BsCM) using quantum mechanics/molecular mechanics (QM/MM) simulation methods. In gas phase, both reactions are described as concerted processes, with the activation free energy for carbachorismate being about 10-15 kcal mol(-)(1) larger than for chorismate, at the AM1 and B3LYP/6-31G levels. Aqueous solution and BsCM active site environments reduce the free energy barriers for both reactions, due to the fact that in these media the two carboxylate groups can be approached more easily than in the gas phase. The enzyme specifically reduces the activation free energy of the Claisen rearrangement about 3 kcal mol(-)(1) more than that for the Cope reaction. This result is due to a larger transition state stabilization associated to the formation of a hydrogen bond between Arg90 and the ether oxygen. When this oxygen atom is changed by a methylene group, the interaction is lost and Arg90 moves inside the active site establishing stronger interactions with one of the carboxylate groups. This fact yields a more intense rearrangement of the substrate structure. Comparing two reactions in the same enzyme, we have been able to obtain conclusions about the relative magnitude of the substrate preorganization and transition state stabilization effects. Transition state stabilization seems to be the dominant effect in this case.