Chorismate active site

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. 

The second part of the reaction requires pyridoxal phosphate (Fig. 22-18). Indole formed in the first part is not released by the enzyme, but instead moves through a channel from the a-subunit active site to the jS-subunit active site, where it condenses with a Schiff base intermediate derived from serine and PLP. Intermediate channeling of this type may be a feature of the entire pathway from chorismate to tryptophan. Enzyme active sites catalyzing different steps (sometimes not sequential steps) of the pathway to tryptophan are found on single polypeptides in some species of fungi and bacte-... 

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... 

Marti S, J Andres, V Moliner, E Silla, I Tunon, J Bertran (2000) A QM/MM study of the conformational equilibria in die chorismate mutase active site. The role of the enzymatic deformation energy contribution. J. Phys. Chem. B 104 (47) 11308—11315... 

Guo H, Q Cui, WN Lipscomb, M Karplus (2001) Substrate conformational transitions in the active site of chorismate mutase Their role in the catalytic mechanism. Proc. Natl. Acad. Sci. U. S. A. 98 (16) 9032-9037... 

Kinetic characterization of several selected BsCM variants shows that truncation or mutation of the C-terminal tail has little effect on the turnover number (fcc ll) of the enzyme (Tab. 3.1). When chorismate is bound to the active site of the variants, it is converted to prephenate nearly as efficiently as with wild-type BsCM. However, a substantial reduction in the k /K value is evident (Tab. 3.1). This finding indicates that the C-terminus, while not directly involved in the chemical transformation of bound ligand, does contribute to enzymatic efficiency by uniform binding of substrate and transition state. 

A thermostable dimer was therefore considered as an alternative starting point for the design of a stable monomeric mutase. A large number of EcCM sequence homo-logues, some from thermophilic organisms, are known. For example, the hyperther-mophilic archaeon Methanococcus jannaschii produces a chorismate mutase (MjCM) that is 25 °C more stable than EcCM [37]. Despite only 21 % sequence identity, six prominent residues that line the active site are strictly conserved and the two enzymes have comparable activities. Since the hydrophobic core of MjCM is very similar to that of EcCM, interactions distant from the dimer interface must be responsible for its additional stability. These same interactions were expected to stabilize the desired monomer. 

To explore the feasibility of such an approach for the design of active catalysts, we have systematically replaced the secondary structural elements in the homodimeric helical bundle chorismate mutase (Fig. 3.18) with binary-patterned units of random sequence. Genetic selection was then used to assess the catalytic capabilities of the proteins in the resulting libraries, providing quantitative information about the robustness of this particular protein scaffold and insight into the subtle interactions needed to form a functional active site [119]. 

COMT is, for many of the same reasons as with chorismate mutase, well suited for the study with computational techniques. The reaction mechanism it catalyzes is the same mechanism that operates in the absence of the enzyme, specifically, the S 2 mechanism, facilitating comparison of the bare solution-phase reaction with the catalyzed reaction. The subsfiate and cofactor do not covalendy bind to the enzyme, so that defining the QM region and the MM region should be relatively uncomplicated. Lasdy, the X-ray crystal structure of COMT bound with the inhibitor 3,5-dinitrocatechol has been determined with a resolution of 2 kP An interesting twist to this enzyme is that the active site includes a metal cation, Mg " ". This crystal structure allows for a natural starting point for computational exploration of the means of the catalytic action of COMT. The rate acceleration provided by COMT is substantial the reaction is 10 times faster within the enzyme than in solution. " ... 

Zhai Z. and Bruice T.C., Temperature dependence of the structure of the substrate and active site of the Thermus thermophilus chorismate mutase E S complex, Biochemistry 45,8562-8567, 2006... 

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.

A mechanistic proposal is outlined in Fig. 16. It is not known whether the car-binolamine 82 or free ammonia reacts with chorismate [69], or whether the addition of the amine involves the initial addition of an active site nucleophile to chorismate [70-74]. 

In another study, the promiscuous chorismate mutase activity of isochorismate pyruvate-lyase (PchB) was used to derive mechanistic insights into its native activity (isochorismate pyruvate lyase). Presumed key active-site residues were randomized, and the resulting variants of PchB were selected for the promiscuous chorismate mutase activity. Consequently, a common mechanism was proposed for both functions of PchB, with the rare [l,5]-sigmatropic rearrangement for the lyase activity, being distinct from other pyruvate lyases. 

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