Chorismate mutase, rearrangement

Chorismate Mutase catalyzed Claisen Rearrangement- 10 rate enhancement over non-enzymatic reaction... 

The enzyme chorismate mutase was found to accelerate the Claisen rearrangement of chorismic acid.147 For many years, the origin of the acceleration perplexed and intrigued chemists and biochemists. Polar... 

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. 

In this contribution, we describe work from our group in the development and application of alternatives that allow the explicit inclusion of environment effects while treating the most relevant part of the system with full quantum mechanics. The first methodology, dubbed MD/QM, was used for the study of the electronic spectrum of prephenate dianion in solution [18] and later coupled to the Effective Fragment Potential (EFP) [19] to the study of the Claisen rearrangement reaction from chorismate to prephenate catalyzed by the chorismate mutase (CM) enzyme [20]. 

The Shikimate pathway is responsible for biosynthesis of aromatic amino acids in bacteria, fungi and plants [28], and the absence of this pathway in mammals makes it an interesting target for designing novel antibiotics, fungicides and herbicides. After the production of chorismate the pathway branches and, via specific internal pathways, the chorismate intermediate is converted to the three aromatic amino acids, in addition to a number of other aromatic compounds [29], The enzyme chorismate mutase (CM) is a key enzyme responsible for the Claisen rearrangement of chorismate to prephenate (Scheme 1-1), the first step in the branch that ultimately leads to production of tyrosine and phenylalanine. 

Besides the obvious biological interest, chorismate mutase is important for being a rare example of an enzyme that catalyses a pericyclic reaction (the Claisen rearrangement), which also occurs in solution without the enzyme, providing a unique... 

Chorismate mutase catalyzes the Claisen rearrangement of chorismate to prephenate at a rate 106 times greater than that in solution (Fig. 5.5). This enzyme reaction has attracted the attention of computational (bio)chemists, because it is a rare example of an enzyme-catalyzed pericyclic reaction. Several research groups have studied the mechanism of this enzyme by use of QM/MM methods [76-78], It has also been studied with the effective fragment potential (EFP) method [79, 80]. In this method the chemically active part of an enzyme is treated by use of the ab initio QM method and the rest of the system (protein environment) by effective fragment potentials. These potentials account... 

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). 

Claisen rearrangement plays an important part in the biosynthesis of several natural products. For example, the chorismate ion is rearranged to the prephenate ion by the Claisen rearrangement, which is catalysed by the enzyme chorismate mutase. This prephenate ion is a key intermediate in the shikimic acid pathway for the biosynthesis of phenylalanine, tyrosine and many other biologically important natural products. 

Chorismate mutase (CM) catalyzes conversion of chorismate into prephenate in a Claisen rearrangement prephenate is a precursor in the biosynthesis of both i-phenylalanine (i-Phe) and i-tyrosine (i-Ttyr). CM occurs as a dimer for structural studies, the monomer was needed (MacBeath, 1998). The assay for CM activity was based on growth of the colonies in the absence of i-Phe and L-Tyr and thus was based on selection, not screening. One resulting mutant was found to be a monomer and to contain a somewhat polar Ala-Arg-Trp-Pro-Trp-Ala sequence. 

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... 

Chorismate mutase catalyses the Claisen rearrangement of chorismate to form prephenate. It is an excellent system for analysing catalysis because the same reaction occurs in solution with the same reaction mechanism no covalent catalysis by the... 

Woodcock HL, M Hodoscek, P Sherwood, YS Lee, HF Schaefer, BR Brooks (2003) Exploring the quantum mechanical/molecular mechanical replica path method a pathway optimization of the chorismate to prephenate Claisen rearrangement catalyzed by chorismate mutase. Theor. Chem. Acc. 109 (3) 140-148... 

The utilization of evolutionary strategies in the laboratory can be illustrated with proteins that catalyze simple metabolic reactions. One of the simplest such reactions is the conversion of chorismate to prephenate (Fig. 3.3), a [3,3]-sigmatropic rearrangement. This transformation is a key step in the shikimate pathway leading to aromatic amino acids in plants and lower organisms [28, 29]. It is accelerated more than a million-fold by enzymes called chorismate mutases [30],... 

Chorismate mutase catalyzes the conversion of chorismate to prephenate (Equation 17.44). This reaction is unusual, in that it is the only pericyclic [3,3] sigmatropic rearrangement (Claisen rearrangement) that is catalyzed by an enzyme. 

Chorismate mutase provides an example of an enzyme where QM/MM calculations have identified an important catalytic principle at work [8], This enzyme catalyses the Claisen rearrangement of chorismate to prephenate. The reaction within the enzyme is not believed to involve chemical catalysis, and this pericylic reaction also occurs readily in solution. Lyne et al. [8] investigated the reaction in chorismate mutase in QM/MM calculations, at the AMI QM level (AMI was found to perform acceptably well for this reaction in comparisons with ab initio results for the reaction in the gas phase [8]). Different sizes of QM system were tested in the QM/MM studies (e.g. including the substrate and no, or up to three, protein side chains), and similar results found in all cases. The reaction was modelled by minimization along an approximate reaction coordinate, defined as the ratio of the forming C-C and breaking C-0 bonds. Values of the reaction coordinate were taken from the AMI intrinsic reaction coordinate for the gas-phase reaction. 

Figure 1 In a QM/MM calculation, a small region is treated by a quantum mechanical (QM) electronic structure method, and the surroundings treated by simpler, empirical, molecular mechanics. In treating an enzyme-catalysed reaction, the QM region includes the reactive groups, with the bulk of the protein and solvent environment included by molecular mechanics. Here, the approximate transition state for the Claisen rearrangement of chorismate to prephenate (catalysed by the enzyme chorismate mutase) is shown. This was calculated at the RHF(6-31G(d)-CHARMM QM-MM level. The QM region here (the substrate only) is shown by thick tubes, with some important active site residues (treated by MM) also shown. The whole model was based on a 25 A sphere around the active site, and contained 4211 protein atoms, 24 atoms of the substrate and 947 water molecules (including 144 water molecules observed by X-ray crystallography), a total of 7076 atoms. The results showed specific transition state stabilization by the enzyme. Comparison with the same reaction in solution showed that transition state stabilization is important in catalysis by chorismate mutase78.

The Claisen rearrangement of chorismate to prephenate, catalyzed by chorismate mutase, is illustrated below. It... 

Catalytic antibodies have been obtained for sigmatropic rearrangements, as first demonstrated for the biotransformation of chorismate to prephenate by two chorismate mutase antibodies developed in parallel by Hilvert et al. and Schultz et al. [76]. The Schultz group also described catalysis of an oxy-Cope rearrangement [77]. Schultz et al. and Hilvert et al. have prepared catalytic antibodies for concerted eUminations of aminoxide 53 and selenoxide 54 from immunization... 

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