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.     

Design and synthesis of aromatic inhibitors of anthranilate synthase

Anthranilate synthase catalyses the conversion of chorismate to anthranilate, a key step in tryptophan biosynthesis. A series of 3-(1-carboxy-ethoxy) benzoic acids were synthesised as chorismate analogues, with varying functionality at C-4, the position of the departing hydroxyl group in chorismate. Most of the compounds were moderate inhibitors of anthranilate synthase, with inhibition constants between 20-30 microM. The exception was 3-(1-carboxy-ethoxy) benzoic acid, (C-4 = H), for which K(I)= 2.4 microM. These results suggest that a hydrogen bonding interaction with the active site general acid (Glu309) is less important than previously assumed for inhibition of the enzyme by these aromatic chorismate analogues.     

Design and synthesis of aromatic inhibitors of anthranilate synthase

Aromatic analogues of chorismate were synthesised as potential inhibitors of anthranilate synthase. Molecular modelling using GOLD2.1 showed that these analogues docked into the active site of Serratia marcescens anthranilate synthase in the same conformation as chorismate. Most compounds were found to be micromolar inhibitors of S. marcescens anthranilate synthase. The most potent analogue, 3-(1-carboxy-ethoxy)-4-hydroxybenzoate (K(I) 3 microM), included a lactyl ether side chain. This appears to be a good replacement for the enol-pyruvyl side chain of chorismate.     

Investigation of the enzymatic and nonenzymatic cope rearrangement of carbaprephenate to carbachorismate

The dimethyl esters of carbaprephenate and 4-epi-carbaprephenate were prepared by modification of published procedures. In methanol these compounds are converted quantitatively to isomeric 6-hydroxytricyclo[3.3.1.0(2,7)]non-3-en-1,3-dimethyl esters via a two-step sequence involving an initial Cope rearrangement, followed by intramolecular Diels-Alder reaction of the dimethyl carbachorismate or 4-epi-carbachorismate intermediates. Carbaprephenate and its epimer were obtained by alkaline hydrolysis of the corresponding dimethyl esters. These compounds, in contrast to their ester precursors, undergo spontaneous acid-catalyzed decarboxylation in aqueous solution. Only at high pH does the Cope rearrangement compete with decarboxylation. At pH 12 and 90 degrees C, carbaprephenate slowly rearranges to carbachorismate, which rapidly loses water to give 3-(2-carboxyallyl)benzoic acid as the major product. A small amount of the intramolecular Diels-Alder adduct derived from carbachorismate is also observed by NMR as a minor product. Carbaprephenate is not a substrate for the enzyme chorismate mutase from Bacillus subtilis (BsCM), nor does carbaprephenate inhibit the normal chorismate mutase activity of this enzyme, even when present in 200-fold excess over chorismate. Its low affinity for the enzyme-active site is presumably a consequence of placing a methylene group rather than an oxygen atom proximal to the essential cationic residue Arg90. Nevertheless, BsCM variants that lack this cation (R90G and R90A) do not accelerate the Cope rearrangement of carbaprephenate either, and a catalytic antibody 1F7, which exhibits modest chorismate mutase activity, is similarly inactive. Poor substrate binding and the relatively high barrier for the Cope compared to the Claisen rearrangement presumably account for the lack of detectable catalysis. Acceleration of this sigmatropic rearrangement apparently requires more than an active site that is complementary in shape to the reactive substrate conformer.     

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.     

Conservation of mechanism in three chorismate-utilizing enzymes

Chorismate is the end-product of the shikimate pathway for biosynthesis of carbocyclic aromatic compounds in plants, bacteria, fungi, and some parasites. Anthranilate synthase (AS), 4-amino-4-deoxychorismate synthase (ADCS), and isochorismate synthase (IS) are homologous enzymes that carry out the initial transformations on chorismate in the biosynthesis of tryptophan, p-aminobenzoate, and enterobactin, respectively, and are expected to share a common mechanism. Poor binding to ADCS of two potential transition state analogues for addition of a nucleophile to C6 of chorismate implies that it, like AS and IS, initiates reaction by addition of a nucleophile to C2. Molecular modeling based on the X-ray structures of AS and ADCS suggests that the active site residue K274 is the nucleophile employed by ADCS to initiate the reaction, forming a covalent intermediate. The K274A and K274R mutants were shown to have 265- and 640-fold reduced k(cat) values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH(4)(+), ADCS and the K274A mutant have identical k(cat) values, suggesting the participation of NH(4)(+) as a rescue agent. Such participation was confirmed by the buildup of 2-amino-2-deoxyisochorismate in the reactions of the K274A mutant but not ADCS, when either NH(4)(+) or PabA + glutamine is used as the nitrogen source. Additionally, the inclusion of ethylamine in the reactions of K274A yields the N-ethyl derivative of 2-amino-2-deoxyisochorismate. A unifying mechanism for AS, ADCS, and IS entailing nucleophile addition to C2 of chorismate in an S(N)2' ' process is proposed.     

A hybrid potential reaction path and free energy study of the chorismate mutase reaction.

We present a combination of two techniques--QM/MM statistical simulation methods and QM/MM internal energy minimizations--to get a deeper insight into the reaction catalyzed by the enzyme chorismate mutase. Structures, internal energies and free energies, taken from the paths of the reaction in solution and in the enzyme have been analyzed in order to estimate the relative importance of the reorganization and preorganization effects. The results we obtain for this reaction are in good agreement with experiment and show that chorismate mutase achieves its catalytic efficiency in two ways; first, it preferentially binds the active conformer of the substrate and, second, it reduces the free energy of activation for the reaction relative to that in solution by providing an environment which stabilizes the transition state.     

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.     

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.     

Inhibition studies on salicylate synthase

Analogues of chorismate and isochorismate were designed and tested as potential inhibitors in the first inhibition study against a salicylate synthase.     

Isotope effects on the enzymatic and nonenzymatic reactions of chorismate

The important biosynthetic intermediate chorismate reacts thermally by two competitive pathways, one leading to 4-hydroxybenzoate via elimination of the enolpyruvyl side chain, and the other to prephenate by a facile Claisen rearrangement. Measurements with isotopically labeled chorismate derivatives indicate that both are concerted sigmatropic processes, controlled by the orientation of the enolpyruvyl group. In the elimination reaction of [4-2H]chorismate, roughly 60% of the label was found in pyruvate after 3 h at 60 degrees C. Moreover, a 1.846 +/- 0.057 2H isotope effect for the transferred hydrogen atom and a 1.0374 +/- 0.0005 18O isotope effect for the ether oxygen show that the transition state for this process is highly asymmetric, with hydrogen atom transfer from C4 to C9 significantly less advanced than C-O bond cleavage. In the competing Claisen rearrangement, a very large 18O isotope effect at the bond-breaking position (1.0482 +/- 0.0005) and a smaller 13C isotope effect at the bond-making position (1.0118 +/- 0.0004) were determined. Isotope effects of similar magnitude characterized the transformations catalyzed by evolutionarily unrelated chorismate mutases from Escherichia coli and Bacillus subtilis. The enzymatic reactions, like their solution counterpart, are thus concerted [3,3]-sigmatropic processes in which C-C bond formation lags behind C-O bond cleavage. However, as substantially larger 18O and smaller 13C isotope effects were observed for a mutant enzyme in which chemistry is fully rate determining, the ionic active site may favor a somewhat more polarized transition state than that seen in solution.     

Quantitative evaluation of noncovalent chorismate mutase-inhibitor binding by ESI-MS

Electrospray time-of-flight mass spectrometry was used to quantitatively determine the dissociation constant of chorismate mutase and a transition state analogue inhibitor. This system presents a fairly complex stoichiometry because the native protein is a homotrimer with three equal and independent substrate binding sites. We can detect the chorismate mutase trimer as well as chorismate mutase-inhibitor complexes by choosing appropriate conditions in the ESI source. To verify that the protein-inhibitor complexes are specific, titration experiments with different enzyme variants and different inhibitors were performed. A plot of the number of bound inhibitors versus added inhibitor concentration revealed saturation behavior with 3:1 (inhibitor:functional trimer) stoichiometry for the TSA. The soft ESI conditions, the relatively high protein mass of 43.5 kDa, and the low charge state (high m/z) result in broad peaks, a typical problem in analyzing noncovalent protein complexes. Due to the low molecular weight of the TSA (226 Da) the peaks of the free protein and the protein with one, two or three inhibitors bound cannot be clearly resolved. For data analysis, relative peak areas of the deconvoluted spectra of chorismate mutase-inhibitor complexes were obtained by fitting appropriate peak shapes to the signals corresponding to the free enzyme and its complexes with one, two, or three inhibitor molecules. From the relative peak areas we were able to calculate a dissociation constant that agreed well with known solution-phase data. This method may be generally useful for interpreting mass spectra of noncovalent complexes that exhibit broad peaks in the high m/z range.     

Charge optimization increases the potency and selectivity of a chorismate mutase inhibitor

The highest affinity inhibitor for chorismate mutases, a conformationally constrained oxabicyclic dicarboxylate transition state analogue, was modified as suggested by computational charge optimization methods. As predicted, replacement of the C10 carboxylate in this molecule with a nitro group yields an even more potent inhibitor of a chorismate mutase from Bacillus subtilis (BsCM), but the magnitude of the improvement (roughly 3-fold, corresponding to a DeltaDeltaG of -0.7 kcal/mol) is substantially lower than the gain of 2-3 kcal/mol binding free energy anticipated for the reduced desolvation penalty upon binding. Experiments with a truncated version of the enzyme show that the flexible C terminus, which was only partially resolved in the crystal structure and hence omitted from the calculations, provides favorable interactions with the C10 group that partially compensate for its desolvation. Although truncation diminishes the affinity of the enzyme for both inhibitors, the nitro derivative binds 1.7 kcal/mol more tightly than the dicarboxylate, in reasonable agreement with the calculations. Significantly, substitution of the C10 carboxylate with a nitro group also enhances the selectivity of inhibition of BsCM relative to a chorismate mutase from Escherichia coli (EcCM), which has a completely different fold and binding pocket, by 10-fold. These results experimentally verify the utility of charge optimization methods for improving interactions between proteins and low-molecular weight ligands.     

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.     

Studies on a new biosynthetic pathway for menaquinone

Menaquinone is biosynthesized from chorismate via a new pathway involving 1,4-dihydroxy-6-naphthoate in Streptomyces and presumably in pathogenic bacteria Helicobacter and Campylobacter.     

Investigation of solvent effects for the Claisen rearrangement of chorismate to prephenate: mechanistic interpretation via near attack conformations

Solvent effects on the rate of the Claisen rearrangement of chorismate to prephenate have been examined in water and methanol. The preequilibrium free-energy differences between diaxial and diequatorial conformers of chorismate, which had previously been implicated as the sole basis for the observed 100-fold rate increase in water over methanol, have been reframed using the near attack conformation (NAC) concept of Bruice and co-workers. Using a combined QM/MM Monte Carlo/free-energy perturbation (MC/FEP) method, 82%, 57%, and 1% of chorismate conformers were found to be NAC structures (NACs) in water, methanol, and the gas phase, respectively. As a consequence, the conversion of non-NACs to NACs provides no free-energy contributions to the overall relative reaction rates in water versus methanol. Free-energy perturbation calculations yielded differences in free energies of activation for the two polar protic solvents and the gas phase. The rate enhancement in water over the gas phase arises from preferential hydration of the transition state (TS) relative to the reactants via increased hydrogen bonding and long-range electrostatic interactions, which accompany bringing the two negatively charged carboxylates into closer proximity. More specifically, there is an increase of 1.3 and 0.6 hydrogen bonds to the carboxylate groups and the ether oxygen, respectively, in going from the reactant to the TS in water. In methanol, the corresponding changes in hydrogen bonding with first shell solvent molecules are small; the rate enhancement arises primarily from the enhanced long-range interactions with solvent molecules. Thus, the reaction occurs faster in water than in methanol due to greater stabilization of the TS in water by specific interactions with first shell solvent molecules.     

Entropic and enthalpic components of catalysis in the mutase and lyase activities of Pseudomonas aeruginosa PchB

The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions, demonstrating the eponymous activity and also chorismate mutase activity. The thermodynamic parameters for these enzyme-catalyzed activities, as well as the uncatalyzed isochorismate decomposition, are reported from temperature dependence of k(cat) and k(uncat) data. The entropic effects do not contribute to enzyme catalysis as expected from previously reported chorismate mutase data. Indeed, an entropic penalty for the enzyme-catalyzed mutase reaction (ΔS(++) = -12.1 ± 0.6 cal/(mol K)) is comparable to that of the previously reported uncatalyzed reaction, whereas that of the enzyme-catalyzed lyase reaction (ΔS(++) = -24.3 ± 0.2 cal/(mol K)) is larger than that of the uncatalyzed lyase reaction (-15.77 ± 0.02 cal/(mol K)) documented here. With the assumption that chemistry is rate-limiting, we propose that a reactive substrate conformation is formed upon loop closure of the active site and that ordering of the loop contributes to the entropic penalty for converting the enzyme substrate complex to the 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.     

Comparison of formation of reactive conformers (NACs) for the Claisen rearrangement of chorismate to prephenate in water and in the E. coli mutase: the efficiency of the enzyme catalysis

The Claisen rearrangements of chorismate (CHOR) in water and at the active site of E. coli chorismate mutase (EcCM) have been compared. From a total of 33 ns molecular dynamics simulation of chorismate in water solvent, seven diaxial conformers I-VII were identified. Most of the time (approximately 99%), the side chain carboxylate of the chorismate is positioned away from the ring due to the electrostatic repulsion from the carboxylate in the ring. Proximity of the two carboxylates, as seen in conformer I, is a requirement for the formation of a near attack conformer (NAC) that can proceed to the transition state (TS). In the EcCM.CHOR complex, the two carboxylates of CHOR are tightly held by Arg28 of one subunit and Arg11* of the other subunit, resulting in the side chain C16 being positioned adjacent to C5 with their motions restricted by van der Waals contacts with methyl groups of Val35 and Ile81. With the definition of NAC as the C5...C16 distance < or =3.7 A and the attack angle < or =30 degrees, it was estimated from our MD trajectories that the free energy of NAC formation is approximately 8.4 kcal/mol above the total ground state in water, whereas in the enzyme it is only 0.6 kcal/mol above the average of the Michaelis complex EcCM.CHOR. The experimentally measured difference in the activation free energies of the water and enzymatic reactions (Delta Delta G(++)) is 9 kcal/mol. It follows that the efficiency of formation of NAC (7.8 kcal/mol) at the active site provides approximately 90% of the kinetic advantage of the enzymatic reaction as compared to the water reaction. Comparison of the EcCM.TSA (transition state analogue) and EcCM.NAC simulations suggests that the experimentally measured 100 fold tighter binding of TSA compared to CHOR does not originate from the difference between NAC and the TS binding affinities, but might be due to the free energy cost to bring the two carboxylates of CHOR together to interact with Arg28 and Arg11* at the active site. The two carboxylates of TSA are fixed by a bicyclic structure. The remaining approximately 10% of Delta Delta G(++) may be attributed to a preferential interaction of Lys39-NH(3)(+) with O13 ether oxygen in the TS.