The catalytic reaction of chorismate mutase (CM) has been the subject of major current attention. Nevertheless, the origin of the catalytic power of CM remains an open question. In particular, it has not been clear whether the enzyme works by providing electrostatic transition state stabilization (TSS), by applying steric strain, or by populating near attack conformation (NAC). The present work explores this issue by a systematic quantitative analysis. The overall catalytic effect is reproduced by the empirical valence bond (EVB) method. In addition, the binding free energy of the ground state and the transition state is evaluated, demonstrating that the enzyme works by TSS. Furthermore, the evaluation of the electrostatic contribution to the reduction of the activation energy establishes that the TSS results from electrostatic effects. It is also found that the apparent NAC effect is not the reason for the catalytic effect but the result of the TSS. It is concluded that in CM as in other enzymes the key catalytic effect is electrostatic TSS. However, since the charge distribution of the transition state and the reactant state is similar, the stabilization of the transition state leads to reduction in the distance between the reacting atoms in the reactant state.

The steric effect, exerted by enzymes on their reacting substrates, has been considered as a major factor in enzyme catalysis. In particular, it has been proposed that enzymes catalyze their reactions by pushing their reacting fragments to a catalytic configuration which is sometimes called near attack configuration (NAC). This work uses computer simulation approaches to determine the relative importance of the steric contribution to enzyme catalysis. The steric proposal is expressed in terms of well defined thermodynamic cycles that compare the reaction in the enzyme to the corresponding reaction in water. The S N2 reaction of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, which was used in previous studies to support the strain concept is chosen as a test case for this proposal. The empirical valence bond (EVB) method provides the reaction potential surfaces in our studies. The reliability and efficiency of this method make it possible to obtain stable results for the steric free energy. Two independent strategies are used to evaluate the actual magnitude of the steric effect. The first applies restraints on the substrate coordinates in water in a way that mimics the steric effect of the protein active site. These restraints are then released and the free energy associated with the release process provides the desired estimate of the steric effect. The second approach eliminates the electrostatic interactions between the substrate and the surrounding in the enzyme and in water, and compares the corresponding reaction profiles. The difference between the resulting profiles provides a direct estimate of the nonelectrostatic contribution to catalysis and the corresponding steric effect. It is found that the nonelectrostatic contribution is about -0.7 kcal/mol while the full "apparent steric contribution" is about -2.2 kcal/mol. The apparent steric effect includes about -1.5 kcal/mol electrostatic contribution. The total electrostatic contribution is found to account for almost all the observed catalytic effect (∼-6.1 kcal/mol of the -6.8 calculated total catalytic effect). Thus, it is concluded that the steric effect is not the major source of the catalytic power of haloalkane dehalogenase. Furthermore, it is found that the largest component of the apparent steric effect is associated with the solvent reorganization energy. This solvent-induced effect is quite different from the traditional picture of balance between the repulsive interaction of the reactive fragments and the steric force of the protein.

The remarkable rate enhancement of orotidine 5'-phosphate decarboxylase (ODCase) has been attributed to ground-state destabilization (GSD) by desolvation and more recently to GSD by electrostatic stress. Here we reiterate our previous arguments that the GSD mechanisms are not likely to play a major role in enzyme catalysis and analyze quantitatively the origin of the rate enhancement of ODCase. This analysis involves energy considerations and computer simulations. Our energy considerations show that (i) the previously proposed desolvation mechanism is based on an improper reference state; (ii) a nonpolar active site cannot account for the catalytic effect of the enzyme; (iii) the focus on the role of the negatively charged protein residues in the electrostatic stress GSD mechanism overlooks the fact that the positively charged Lys72 strongly stabilizes the substrate; (iv) although the previous calculation of the actual enzymatic reaction correctly reproduced the observed rate enhancement, it could not obtain this rate enhancement from the calculated binding energies (which are the relevant quantities for determining GSD effects); (v) the GSD mechanism is inconsistent with the observed binding energy of the phosphoribosyl part of the substrate; and (vi) the presumably unstable substrate (orotate) can be stabilized, at equilibrium, by accepting a proton from the solvent. Our computer simulation studies involve two set of calculations. First, we study the catalytic reaction by using an empirical valence bond potential surface calibrated by ab initio calculations of the reference solution reaction. This calculation reproduces the observed catalytic effect of the enzyme. Next, we use free-energy perturbation calculations and evaluated the electrostatic contributions to the binding energies of the ground state and transition state (TS). These calculations show that the rate enhancement in ODCase is due to the TS stabilization rather than to GSD. The differences between our own and the previous theoretical analyses stem from both the selection of the reacting system and the treatment of the long-range electrostatic contributions to the binding energy. The reacting system was previously assumed to encompass only the orotate. However, this selection does not allow proper description of the reaction catalyzed by the enzyme (i.e., [Orotate(-) + LysH(+)] if [uracil + Lys + CO(2)]). Therefore, the reacting system should include both orotate and the general acid in the form of the protonated Lys72 protein residue. This selection leads to a simple and consistent interpretation of the catalytic effect where the electrostatic stabilization of the transition state is due to the fact that the two negatively charged aspartic residues are already placed near the reactive lysine so that they do not have to reorganize significantly during the reaction. Interestingly, even calculations with only orotate(-) as the reacting system do not produce sufficient destabilization to account for a GSD mechanism. In summary, we conclude, in agreeme...

Quantum mechanical calculations of activation free energies of chemical reactions in condensed phases present a major challenge for computational chemistry. On one hand, it is important to use high-level ab initio methods to obtain reliable results. On the other hand, it is essential to perform sufficient configurational sampling to obtain meaningful free energies. Although the advance of quantum mechanical/molecular mechanics (QM/ MM) approaches has made this problem tractable, it still requires an enormous amount of computer time. The present work advances several strategies that allow one to perform practical ab initio QM/MM calculations of free energy profiles in solutions and proteins. The basic idea is the use of a simple reference potential for the ab initio calculations (e.g., Bentzien; et al. J. Phys. Chem. B 1998, 102, 2293. One version of this approach evaluates the free energy of transfer from the reference potential to the ab initio potential by a single step free energy perturbation (FEP) approach. A new version evaluates this free energy by the linear response approximation (LRA), which involves running trajectories on both the reference and the ab initio potentials. The performance of both approaches is examined by calculating the potential of mean force for the autodissociation reaction of water in solution. It is found that the LRA approach allows one to obtain reasonable results even in cases where the ab initio and reference potentials are significantly different. The present work also explores options for increasing the size of the quantum mechanical region. Here it is shown that the constrained DFT (CDFT) method provides a promising strategy. Finally, the general issue of modeling the autodissociation reaction by quantum mechanical approaches is briefly considered. It is pointed out that the use of the empirical valence bond (EVB) approach in the sampling process should provide a way for evaluating the elusive nonequilibrium solvation effect.

To elucidate the catalytic power of enzymes it is crucial to have clear information about the corresponding reference reactions in solution. This is needed since catalysis is defined by comparing enzymatic reactions to the relevant uncatalyzed reactions. Unfortunately, the energetics of the reference reactions of many important classes of enzymatic reactions have not been fully determined by experimental studies. In many cases it is hard to determine whether the given reaction involves a stepwise or a concerted mechanism. It is also hard to estimate the activation barrier for steps which are not rate determining. Fortunately, it is possible now to use computational approaches to augment the available experiments and to elucidate the shape of free energy surfaces of various reference reactions. Here we present a systematic study of the reference solution reaction for studies of serine proteases, i.e., the base-catalyzed and general base/acid catalyzed methanolysis of formamide. The present work is based on the use of combined ab initio/Langevin dipoles calculations and on a careful comparison to available experiments. The applied ab initio methodologies involve nonlocal density functional (B3LYP/AUG-cc-pVDZ) calculations and G2 theory. The construction of the relevant free energy surfaces involves partial geometry optimizations for the ammonia-catalyzed methanolysis of formamide. Subsequently, energy corrections based on the appropriate experimental pK a values are applied to construct interpolated free energy surfaces for the water- and histidine-catalyzed reactions. Crucial points on the free energy surface for the water catalyzed reaction are also evaluated independently. We start by exploring the first step of the alcoholysis reaction, which involves a proton transfer from ROH to a base, a nucleophilic attack of RO- on the amide, and a formation of a tetrahedral intermediate anion (TI). The interpolated free energy surface for the water-assisted alcoholysis involves a least energy path where the proton transfer is concerted with the nucleophilic attack. The corresponding activation barrier is ∼32 kcal/mol. The independently calculated surface for this reaction involves an activation barrier of ∼34 kcal/mol. These results are in a good agreement with the corresponding experimentally observed barrier (30−32 kcal/mol). The interpolated free energy surface for the histidine-catalyzed reaction involves a stepwise path, with a shallow surface that can also allow for a concerted path. This free energy surface is quite different than the fully concerted surface obtained in previous theoretical studies. The calculated activation barrier for the hisitidine-catalyzed reaction is around 26 kcal/mol. To examine the next step of the reaction we evaluated the basicities of the O and N atoms of the TI. These values were found to be 14 and 8 pK a units, respectively. The calculated pK a of the N atom indicates that the leaving group is protonated prior to the cleavage of the CN bond. The activation free energy for the CN bond ...