Chorismate transition state

The differences in the rate constant for the water reaction and the catalyzed reactions reside in the mole fraction of substrate present as near attack conformers (NACs).171 These results and knowledge of the importance of transition-state stabilization in other cases support a proposal that enzymes utilize both NAC and transition-state stabilization in the mix required for the most efficient catalysis. 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.172 The fact that the reaction occurred faster in water than in methanol was attributed to greater stabilization of the TS in water by specific interactions with first-shell solvent molecules. The Claisen rearrangements of chorismate in water and at the active site of E. coli chorismate mutase have been compared.173 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 with the water reaction. 

Scheme 1-1. Transition state for the conversion of chorismate into prephenate. Also indicated are the Glu78 and Arg90 residues from chorismate mutase...

Schultz and coworkers (Jackson et a ., 1988) have generated an antibody which exhibits behaviour similar to the enzyme chorismate mutase. The enzyme catalyses the conversion of chorismate [49] to prephenate [50] as part of the shikimate pathway for the biosynthesis of aromatic amino acids in plants and micro-organisms (Haslam, 1974 Dixon and Webb, 1979). It is unusual for an enzyme in that it does not seem to employ acid-base chemistry, nucleophilic or electrophilic catalysis, metal ions, or redox chemistry. Rather, it binds the substrate and forces it into the appropriate conformation for reaction and stabilizes the transition state, without using distinct catalytic groups. 

The conversion of [49] into [50] involves a Claisen rearrangement. Once this was realized it was less surprising that no specific catalytic groups on the enzyme are involved. Support for the Claisen-type mechanism comes from the inhibition shown by the bicyclic dicarboxylate [51], prepared by Bartlett and Johnson (1985) as an analogue of the presumed transition state [52], This same structure [51], coupled through the hydroxyl group to a small protein, was used as a hapten to induce antibodies, one (out of eight) of which mimics the behaviour of chorismate mutase, albeit less efficiently (Table 7). 

Figure 11 Chorismate-prephenate rearrangement catalyzed by antibody 1F7 raised against a bicyciic hapten that mimics the chair conformation of the transition state of the reaction and X-ray structure of the corresponding antibody 1 F7-hapten compiex.

Using the transition-state analog shown on p. 485 a catalytic antibody with chorismate mutase activity was isolated. Many antibodies catalyzing additional reactions have also been found. Although they are usually less active than natural enzymes, in some cases they approach enzymatic rates. Furthermore, they may catalyze reactions for which no known enzymes exist.h... 

The reaction, which proceeds via a conformationally tight chair-type transition state, is clearly entropically dominated, with a AS of -13 eu. Whereas the known enzyme chorismate mutase from E. coli achieves a 3 x 106-fold accelerated catalysis, the antibody reaches a 104-fold enhancement. A decrease of AS to almost 0 eu points to the presence of an entropy trap. 

Figure 4.11). This reaction, a Claisen rearrangement, transfers the PEP-derived side-chain so that it becomes directly bonded to the carbocycle, and so builds up the basic carbon skeleton of phenylalanine and tyrosine. The reaction is catalysed in nature by the enzyme chorismate mutase, and, although it can also occur thermally, the rate increases some 106-fold in the presence of the enzyme. The enzyme achieves this by binding the pseudoaxial conformer of chorismic acid, allowing a transition state with chairlike geometry to develop. 

Competitive experiments with 2H-, 13C- and 180-labelled chorismate derivatives and three different chorismate mutase enzymes have shown that in all catalysed and non-catalysed Claisen rearrangements a non-synchronous, concerted, pericyclic transition state is involved, with C-O bond cleavage considerably in advance of C-C bond formation. Some evidence has suggested that the ionic active site of the enzymes may polarize the transition state more than occurs in solution. Similar findings apply to the retro-ene fragmentation of chorismate to 4-hydroxybenzoate.17... 

Scheme 4.12 Catalytic antibody 1F7 was raised against the transition state analog 28 and possesses modest chorismate mutase activity. It can complement a permissive yeast strain that is auxotrophic for phenylalanine and tyrosine by replacingthe natural enzyme (CM) in the shikimate biosynthetic pathway.

Szefczyk B, Mulholland AJ, Ranaghan KE, Sokalski WA (2004) Differential transition-state stabilization in enzyme catalysis Quantum chemical analysis of interactions in the chorismate mutase reaction and prediction of the optimal catalytic field. J Am Chem Soc 126 16148—16159... 

QM/MM methods have proved their value for enzyme reactions in differentiating between alternative proposed mechanisms, and in analysing contributions to catalysis. A current example is the analysis of the contribution of conformational effects and transition state stabilization in the reaction catalysed by the enzyme chorismate mutase.98,99 QM/MM calculations can be performed with... 

Recent investigations of the enzyme chorismate mutase show how modelling can contribute to fundamental debates in enzymology, such as analysing the importance of transition state stabilization in catalysis, and alternative proposals to explain enzyme catalytic proficiency. 

Ranaghan KE, L Ridder, B Szefczyk, WA Sokalski, JC Hermann, AJ Mulholland (2004) Transition state stabilization and substrate strain in enzyme catalysis ab initio QM/MM modelling of the chorismate mutase reaction. Organic Biomolecular Chemistry 2 (7) 968-980... 

Strajbl M, A Shurki, M Kato, A Warshel (2003) Apparent NAC effect in chorismate mutase reflects electrostatic transition state stabilization. J. Am. Chem. Soc. 125 (34) 10228-10237... 

Guimaraes CRW, MP Repasky, J Chandrasekhar, J Tirado-Rives, WL Jorgensen (2003) Contributions of conformational compression and preferential transition state stabilization to the rate enhancement by chorismate mutase. J. Am. Chem. Soc. 125 (23) 6892-6899... 

Claeyssens F, KE Ranaghan, FR Manby, JN Harvey, AJ Mulholland (2005) Multiple high-level QM/MM reaction paths demonstrate transition-state stabilization in chorismate mutase correlation of barrier height with transition-state stabilization. Chem. Comm. (40) 5068—5070... 

Before we embark on our journey into the world of six-membered transition states, I would like to speak briefly about one reaction, to illustrate how a transition state is drawn throughout the book. The enzyme-catalyzed transformation of chorsimate (2) to prephenate (3) is a classic example of a [3,3]-sigmatropic Claisen rearrangement6 (Scheme IV). As an old bond is being broken and at the same time a new bond is formed in the transition state, the transition state for the Claisen rearrangement of chorismate to prephenate would look more like transistion state A than like B. Still, for the convenience of following the bond connection event clearly, I prefer to draw the transition state like B. 

Adamantane has an extra methylene bridge (the asterisk in the diagram) linked to the six-membered ring, thus stabilizing a cagelike structure. The authors indeed subsequently showed that some adamantane derivatives are potent inhibitors of chorismate mutase thus, these are examples of transition-state analogs. 

The most favourable conformation for chorismic acid has the substituents pseudo equatorial but the L3,3]-sigmatropic rearrangement cannot take place in that conformation. First, the diaxial conformation must be formed and the chair transition state achieved. Then the required orbitals will be correctly aligned. 

In order to provide a better estimate of the enantioselectivity of the catalyst, we prepared an authentic sample of (+)-chorismate by kinetic resolution of the racemate with 1F7 (37). Circular dichroism spectroscopy confirmed the identity and high optical purity of the recovered, HPLC-purified compound. Initial rate measurements with the individual isomers show that (-)-chorismate is favored over (+)-chorismate by the antibody by a factor of at least 90 to 1 at low substrate concentrations. The slight rate enhancements above background observed for the (+)-isomer may be due to general medium effects rather than interaction with a specific locus on the antibody surface. To test this possibility we are currently examining the ability of the transition state analog 3 to inhibit rearrangement of this optical isomer. 

The transition state for the enzymatic reaction has been shown to have a chairlike geometry as well [61], and conformationally constrained compounds that mimic this structure, such as the oxabicyclic dicarboxylic acid 1 (Fig. 3.6), are good inhibitors of chorismate mutase enzymes [62 - 64], How a protein might stabilize this high-energy species has been a matter of some debate. Recently, heavy atom isotope effects were used to characterize the structure of the transition state bound to BsCM [65]. A very... 

Fig. 3.6. Chorismate prefers a pseudodiequatorial conformation in solution. It must adopt a disfavored pseudodiaxial conformation to reach the pericyclic transition state. The conformationally constrained oxabicyclic dicarboxylic acid 1, which mimics the transition state, is a potent inhibitor of natural chorismate mutases [62], Antibodies raised against this compound also catalyze the reaction, albeit 100 to 10,000-times less efficiently than their natural counterparts [39, 41].

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