Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor

Citrulline was incorporated via chemical semisynthesis at position 90 in the active site of the AroH chorismate mutase from Bacillus subtilis. The wild-type arginine at this position makes hydrogen-bonding interactions with the ether oxygen of chorismate. Replacement of the positively charged guanidinium group with the isosteric but neutral urea has a dramatic effect on the ability of the enzyme to convert chorismate into prephenate. The Arg90Cit variant exhibits a >104-fold decrease in the catalytic rate constant kcat with a 2.7-fold increase in the Michaelis constant Km. In contrast, its affinity for a conformationally constrained inhibitor molecule that effectively mimics the geometry but not the dissociative character of the transition state is only reduced by a factor of approximately 6. These results show that an active site merely complementary to the reactive conformation of chorismate is insufficient for catalysis of the mutase reaction. Instead, electrostatic stabilization of the polarized transition state by provision of a cationic hydrogen bond donor proximal to the oxygen in the breaking C-O bond is essential for high catalytic efficiency.     

Contributions of conformational compression and preferential transition state stabilization to the rate enhancement by chorismate mutase

The rate enhancement provided by the chorismate mutase (CM) enzyme for the Claisen rearrangement of chorismate to prephenate has been investigated by application of the concept of near attack conformations (NACs). Using a combined QM/MM Monte Carlo/free-energy perturbation (MC/FEP) method, 82% and 100% of chorismate conformers were found to be NAC structures in water and in the CM active site, respectively. Consequently, the conversion of non-NACs to NACs does not contribute to the free energy of activation from preorganization of the substrate into NACs. The FEP calculations yielded differences in free energies of activation that well reproduce the experimental data. Additional calculations indicate that the rate enhancement by CM over the aqueous phase results primarily from conformational compression of NACs by the enzyme and that this process is enthalpically controlled. This suggests that preferential stabilization of the transition state in the enzyme environment relative to water plays a secondary role in the catalysis by CM.     

Transition state stabilization and substrate strain in enzyme catalysis: ab initio QM/MM modelling of the chorismate mutase reaction

To investigate fundamental features of enzyme catalysis, there is a need for high-level calculations capable of modelling crucial, unstable species such as transition states as they are formed within enzymes. We have modelled an important model enzyme reaction, the Claisen rearrangement of chorismate to prephenate in chorismate mutase, by combined ab initio quantum mechanics/molecular mechanics (QM/MM) methods. The best estimates of the potential energy barrier in the enzyme are 7.4-11.0 kcal mol(-1)(MP2/6-31+G(d)//6-31G(d)/CHARMM22) and 12.7-16.1 kcal mol(-1)(B3LYP/6-311+G(2d,p)//6-31G(d)/CHARMM22), comparable to the experimental estimate of Delta H(++)= 12.7 +/- 0.4 kcal mol(-1). The results provide unequivocal evidence of transition state (TS) stabilization by the enzyme, with contributions from residues Arg90, Arg7, and Arg63. Glu78 stabilizes the prephenate product (relative to substrate), and can also stabilize the TS. Examination of the same pathway in solution (with a variety of continuum models), at the same ab initio levels, allows comparison of the catalyzed and uncatalyzed reactions. Calculated barriers in solution are 28.0 kcal mol(-1)(MP2/6-31+G(d)/PCM) and 24.6 kcal mol(-1)(B3LYP/6-311+G(2d,p)/PCM), comparable to the experimental finding of Delta G(++)= 25.4 kcal mol(-1) and consistent with the experimentally-deduced 10(6)-fold rate acceleration by the enzyme. The substrate is found to be significantly distorted in the enzyme, adopting a structure closer to the transition state, although the degree of compression is less than predicted by lower-level calculations. This apparent substrate strain, or compression, is potentially also catalytically relevant. Solution calculations, however, suggest that the catalytic contribution of this compression may be relatively small. Consideration of the same reaction pathway in solution and in the enzyme, involving reaction from a 'near-attack conformer' of the substrate, indicates that adoption of this conformation is not in itself a major contribution to catalysis. Transition state stabilization (by electrostatic interactions, including hydrogen bonds) is found to be central to catalysis by the enzyme. Several hydrogen bonds are observed to shorten at the TS. The active site is clearly complementary to the transition state for the reaction, stabilizing it more than the substrate, so reducing the barrier to reaction.     

A transition path sampling study of the reaction catalyzed by the enzyme chorismate mutase

The study of the chemical steps in enzyme-catalyzed reactions represents a challenge for molecular simulation techniques. One concern is how to calculate paths for the reaction. Common techniques include the definition of a reaction coordinate in terms of a small set of (normally) geometrical variables or the determination of minimum energy paths on the potential energy surface of the reacting system. Both have disadvantages, the former because it presupposes knowledge of which variables are likely to be important for reaction and the latter because it provides a static picture and dynamical effects are ignored. In this paper, we employ the transition path sampling method developed by Chandler and co-workers, which overcomes some of these limitations. The reaction that we have chosen is the chorismate-mutase-catalyzed conversion of chorismate into prephenate, which has become something of a test case for simulation studies of enzyme mechanisms. We generated an ensemble of approximately 1000 independent transition paths for the reaction in the enzyme and another approximately 500 for the corresponding reaction in solution. A large variety of analyses of these paths was performed, but we have concentrated on characterizing the transition state ensemble, particularly the flexibility of its structures with respect to other ligands of the enzyme and the time evolution of various geometrical and energetic properties as the reaction proceeds. We have also devised an approximate technique for locating transition state structures along the paths.     

Multiple high-level QM/MM reaction paths demonstrate transition-state stabilization in chorismate mutase: correlation of barrier height with transition-state stabilization

Multiple profiles for the reaction from chorismate to prephenate in the enzyme chorismate mutase calculated with hybrid density functional combined quantum mechanics/molecular mechanics methods (B3LYP/6-31G(d)-CHARMM27) agree well with experiment, and provide direct evidence of transition-state stabilization by this important enzyme, which is at the centre of current debates about the nature of enzyme catalysis.     

Multiple-steering QM-MM calculation of the free energy profile in chorismate mutase

A novel technique for computing free energy profiles in enzymatic reactions using the multiple steering molecular dynamics approach in the context of an efficient QM-MM density functional scheme is presented. The conversion reaction of chorismate to prephenate catalyzed by the Bacillus subtilis enzyme chorismate mutase has been chosen as an illustrative example.     

Differential transition-state stabilization in enzyme catalysis: quantum chemical analysis of interactions in the chorismate mutase reaction and prediction of the optimal catalytic field

Chorismate mutase is a key model system in the development of theories of enzyme catalysis. To analyze the physical nature of catalytic interactions within the enzyme active site and to estimate the stabilization of the transition state (TS) relative to the substrate (differential transition state stabilization, DTSS), we have carried out nonempirical variation-perturbation analysis of the electrostatic, exchange, delocalization, and correlation interactions of the enzyme-bound substrate and transition-state structures derived from ab initio QM/MM modeling of Bacillus subtilis chorismate mutase. Significant TS stabilization by approximately -23 kcal/mol [MP2/6-31G(d)] relative to the bound substrate is in agreement with that of previous QM/MM modeling and contrasts with suggestions that catalysis by this enzyme arises purely from conformational selection effects. The most important contributions to DTSS come from the residues, Arg90, Arg7, Glu78, a crystallographic water molecule, Arg116, and Arg63, and are dominated by electrostatic effects. Analysis of the differential electrostatic potential of the TS and substrate allows calculation of the catalytic field, predicting the optimal location of charged groups to achieve maximal DTSS. Comparison with the active site of the enzyme from those of several species shows that the positions of charged active site residues correspond closely to the optimal catalytic field, showing that the enzyme has evolved specifically to stabilize the TS relative to the substrate.     

Kinetic isotope effects in the active site of B. subtilis chorismate mutase

Kinetic isotope effects are determined for the enzyme‐catalyzed Claisen rearrangement of chorismate to prephenate using computational methods. The calculated kinetic isotope effects (KIEs) compare reasonably with the few available experimental values with both the theory and experiment obtaining a large KIE for the ether oxygen, indicating large polarization of the transition‐state geometry. Because there is a question of the extent that the experimental rate constants are for chemistry as the rate‐limiting step, the KIEs for all the atoms of the substrate are reported with the exception of the carboxylate groups. A substantial number of large regular and inverse isotope effects are predicted for the hydrogens on the cyclohexadienyl ring related to activation of the reactant and charge reorganization in the transition state. A large KIE is predicted for the hydrogen atom bound to the ether carbon atom because the largest valency change and charge transfer occurs at the ether bond in both the reactant and tansition state. Observation of the overall pattern of predicted KIEs would ensure that conditions are favorable for the rate‐limiting chemistry. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 94: 287–292, 2003     

Preorganization and reorganization as related factors in enzyme catalysis: the chorismate mutase case

In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Bacillus subtilis chorismate mutase, is provided by means of a combination of statistical quantum mechanics/molecular mechanics simulation methods and hybrid potential energy surface exploration techniques. The main aim of this work is to present an estimation of the preorganization and reorganization terms of the enzyme catalytic rate enhancement. To analyze the first of these, we have studied different conformational equilibria of chorismate in aqueous solution and in the enzyme active site. Our conclusion is that chorismate mutase preferentially binds the reactive conformer of the substrate--that presenting a structure similar to the transition state of the reaction to be catalyzed--with shorter distances between the carbon atoms to be bonded and more diaxial character. With respect to the reorganization effect, an energy decomposition analysis of the potential energies of the reactive reactant and of the reaction transition state in aqueous solution and in the enzyme shows that the enzyme structure is better adapted to the transition structure. This means not only a more negative electrostatic interaction energy with the transition state but also a low enzyme deformation contribution to the energy barrier. Our calculations reveal that the structure of the enzyme is responsible for stabilizing the transition state structure of the reaction, with concomitant selection of the reactive form of the reactants. This is, the same enzymatic pattern that stabilizes the transition structure also promotes those reactant structures closer to the transition structure (i.e., the reactive reactants). In fact, both reorganization and preorganization effects have to be considered as the two faces of the same coin, having a common origin in the effect of the enzyme structure on the energy surface of the substrate.     

Influence of diene substitution on transition state stabilization in Diels-Alder reaction

A theoretical analysis of transition state stabilization in D-A reactions of substituted dienes according to the nature and position of the substituent has been carried on. Results revealed that substituents (de)stabilize TS through four effects (steric, mesomeric, inductive, and polarizability) acting principally by favoring the electronic transfer between the two partners. The correlations observed point out nevertheless that the reactivity of substituted dienes in [4 + 2] cycloadditions on ethylene may principally be predicted by the sole use of the F + R electronic parameters.     

QM/MM calculations of kinetic isotope effects in the chorismate mutase active site

Kinetic isotope effects have been computed for the Claisen rearrangement of chorismate to prephenate in aqueous solution and in the active site of chorismate mutase from B. subtilus. These included primary 13C and 18O and secondary 3H effects for substitutions at the bond-making and bond-breaking positions. The initial structures of the putative stationary points on the potential energy surface, required for the calculations of isotope effects using the CAMVIB/CAMISO programs, have been selected from hybrid QM/MM molecular dynamical simulations using the DYNAMO program. Refinement of the reactant complex and transition-state structures has been carried out by means of AM1/CHARMM24/TIP3P calculations using the GRACE program, with full gradient relaxation of the position of > 5200 atoms for the enzymic simulations, and with a box containing 711 water molecules for the corresponding reaction in aqueous solution. Comparison of these results, and of gas phase calculations, with experimental data has shown that the chemical rearrangement is largely rate-determining for the enzyme mechanism. Inclusion of the chorismate conformational pre-equilibrium step in the modelled kinetic scheme leads to better agreement between recent experimental data and theoretical predictions. These results provide new information on an important enzymatic transformation, and the key factors responsible for the kinetics of its molecular mechanism are clarified. Treatment of the enzyme and/or solvent environment by means of a large and flexible model is absolutely essential for prediction of kinetic isotope effects.     

Transition structure selectivity in enzyme catalysis: a QM/MM study of chorismate mutase

Two different transition structures (TSs) have been located and characterized for the chorismate conversion to prephenate in Bacillus subtilis chorismate mutase by means of hybrid quantum-mechanical/molecular-mechanical (QM/MM) calculations. GRACE software, combined with an AM1/CHARMM24/TIP3P potential, has been used involving full gradient relaxation of the position of ca. 3300 atoms. These TSs have been connected with their respective reactants and products by the intrinsic reaction coordinate (IRC) procedure carried out in the presence of the protein environment, thus obtaining for the first time a realistic enzymatic reaction path for this reaction. Similar QM/MM computational schemes have been applied to study the chemical reaction solvated by ca. 500 water molecules. Comparison of these results together with gas phase calculations has allowed understanding of the catalytic efficiency of the protein. The enzyme stabilizes one of the TSs (TS_OHout) by means of specific hydrogen bond interactions, while the other TS (TS_OHin) is the preferred one in vacuum and in water. The enzyme TS is effectively more polarized but less dissociative than the corresponding solvent and gas phase TSs. Electrostatic stabilization and an intramolecular charge-transfer process can explain this enzymatically induced change. Our theoretical results provide new information on an important enzymatic transformation and the key factors responsible for efficient selectivity are clarified. Received: 25 March 2000 / Accepted: 7 August 2000 / Published online: 23 November 2000     

Crowding effect on helix-coil transition: beyond entropic stabilization

We report circular dichroism measurements on the helix-coil transition of poly(L-glutamic acid) in solution with polyethylene glycol (PEG) as a crowding agent. The PEG solutions have been characterized by small angle neutron scattering and are well described by the picture of a network of mesh size ξ, usual for semi-dilute chains in good solvent. We show that the increase of PEG concentration stabilizes the helices and increases the transition temperature. But more unexpectedly, we also notice that the increase of concentration of crowding agent reduces the mean helix extent at the transition, or in other words reduces its cooperativity. This result cannot be taken into account for by an entropic stabilization mechanism. Comparing the mean length of helices at the transition and the mesh size of the PEG network, our results strongly suggest two regimes: helices shorter or longer than the mesh size.     

Sphere-to-cylinder transition in hierarchical electrostatic complexes

We report on the formation of colloidal complexes resulting from the electrostatic co-assembly between anionic surfactants and cationic polyelectrolytes or block copolymers. Combining light and X-ray scattering experiments with cryogenic transmission and optical microscopy, we emphasize a feature rarely addressed in the formation of the electrostatic complexes, namely the role of the mixing concentration on the microstructure. At low mixing concentration, electrostatic complexes made from cationic-neutral copolymers and alkyl sulfate surfactants exhibit spherical core-shell microstructures. With increasing concentration, the complexes undergo a sphere-to-cylinder transition, yielding elongated aggregates with diameter 50 nm and length up to several hundreds of nanometers. From the comparison between homo- and diblock polymer phase behaviors, it is suggested that the unidimensional growth is driven by the ability of the surfactant to self-assemble into cylindrical micelles when complexed with cationic polymers.     

Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design

Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs. Here, we report the synthesis of three carbasugar isotopologues and provide a detailed transition state (TS) analysis for the formation of the initial GH-carbasugar covalent intermediate, as well as for hydrolysis of this intermediate, using a combination of experimentally measured kinetic isotope effects and hybrid QM/MM calculations. We find that the α-galactosidase from Thermotoga maritima effectively stabilizes TS charge development on a remote C5-allylic center acting in concert with the reacting carbasugar, and catalysis proceeds via an exploded, or loose, S_N2 transition state with no discrete enzyme-bound cationic intermediate. We conclude that, in complement to what we know about the TS structures of enzyme-natural substrate complexes, knowledge of the TS structures of enzymes reacting with non-natural carbasugar substrates shows that GHs can stabilize a wider range of positively charged TS structures than previously thought. Furthermore, this enhanced understanding will enable the design of new carbasugar GH transition state analogues to be used as, for example, chemical biology tools and pharmaceutical lead compounds.

Positive charge stabilized on remote C5-allylic center with catalysis occurring via a loose S_N2 transition state.     

Conformational effects in enzyme catalysis: QM/MM free energy calculation of the 'NAC' contribution in chorismate mutase

The controversial 'near attack conformation'(NAC) effect in the important model enzyme chorismate mutase is calculated to be 3.8-4.6 kcal mol(-1) by QM/MM free energy perturbation molecular dynamics methods, showing that the NAC effect by itself does not account for catalysis in this enzyme.     

Exclusive transition state stabilization in the supramolecular catalysis of Diels-Alder reaction by a uranyl salophen complex

Whereas the parent uranyl salophen is catalytically inactive, its phenyl derivative effectively catalyses with turnover the reaction of benzoquinone with 1,3-cyclohexadiene, while showing no appreciable affinity towards reactants and product.     

All electron quantum chemical calculation of the entire enzyme system confirms a collective catalytic device in the chorismate mutase reaction

To elucidate the catalytic power of enzymes, we analyzed the reaction profile of Claisen rearrangement of Bacillus subtilis chorismate mutase (BsCM) by all electron quantum chemical calculations using the fragment molecular orbital (FMO) method. To the best of our knowledge, this is the first report of ab initio-based quantum chemical calculations of the entire enzyme system, where we provide a detailed analysis of the catalytic factors that accomplish transition-state stabilization (TSS). FMO calculations deliver an ab initio-level estimate of the intermolecular interaction between the substrate and the amino acid residues of the enzyme. To clarify the catalytic role of Arg90, we calculated the reaction profile of the wild-type BsCM as well as Lys90 and Cit90 mutant BsCMs. Structural refinement and the reaction path determination were performed at the ab initio QM/MM level, and FMO calculations were applied to the QM/MM refined structures. Comparison between three types of reactions established two collective catalytic factors in the BsCM reaction: (1) the hydrogen bonds connecting the Glu78-Arg90-substrate cooperatively control the stability of TS relative to the ES complex and (2) the positive charge on Arg90 polarizes the substrate in the TS region to gain more electrostatic stabilization.