Chorismate reactions

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

Biological examples of pericyclic reactions are relatively rare, although one much-studied example occurs during biosynthesis in bacteria of the essential amino acid phenylalanine. Phenylalanine arises from the precursor chorismate,... 

Within the diastereomeric switch sequences, the corresponding trans-diols become accessible either using a Mitsunobu inversion or a reversible Diels-Alder cyclization as key reaction step [249,250]. This synthetic strategy is complementary to an approach involving metabolic engineering of E. coli via the chorismate/ isochorismate pathway [251]. 

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]. 

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... 

Figure 1-4. Energy profiles for the reaction of chorismate to prephenate. (a) Profile in vacuum for the forward (squares) and reverse (filled circles) reactions, (b) Profiles for forward reaction in water (filled circles), and in the enzyme with only the substrate in the QM zone (squares) and with substrate plus chorismate mutase side chains glu78 and arg90 in the QM zone (diamonds)...

Figure 1-5. Free energy profile for the reaction from chorismate (RC 1.75) to prephenate (RC — 1.75), obtained using MSMD and Jarzynski s equality and pulling speeds of 2.0 A/ps (red) and 1.0 A/ps (green), and using umbrella sampling (blue)...

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 partitioning of the system in a QM/MM calculation is simpler if it is possible to avoid separating covalently bonded atoms at the border between the QM and the MM regions. An example is the enzyme chorismate mutase [39] for which the QM region could include only the substrate, because the enzyme does not chemically catalyze this pericyclic reaction. In studies of enzyme mechanisms, however, this situation is exceptional, and usually it will be essential, or desirable, to include parts of the protein (for example catalytic residues) in the QM region of a QM/MM calculation, i.e. the boundary between the QM and MM regions will separate covalently bonded atoms (Fig. 6.1). 

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. 

Aryl side chain containing L-a-amino acids, such as phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), are derived through the shikimate pathway. The enzymatic transformation of phosphoenolpyr-uvate (PEP) and erythro-4-phosphate, through a series of reactions, yields shikimate (Scheme 2). Although shikimate is an important biosynthetic intermediate for a number of secondary metabolites, this chapter only describes the conversion of shikimate to amino acids containing aryl side chains. In the second part of the biosynthesis, shikimate is converted into chorismate by the addition of PEP to the hydroxyl group at the C5 position. Chorismate is then transformed into prephenate by the enzyme chorismate mutase (Scheme 3). 

During the biosynthetic transformation, chorismate is the point of divergence for the biosynthesis of Phe, Tyr, Trp, and other amino acids containing aromatic groups. For example, the biosynthesis of Trp begins with the conversion of chorismate to anthranilate (Scheme 4(a)). A sequence of amination and aromatization reactions produces anthranilate, which is then condensed with phosphoribosylpyrophosphate. The intermediate is carried through a series of reactions to yield Trp (Scheme 4(b)). 

The antibodies can also act like entropy traps by stabilizing a particular conformation of a substrate that is favorable to the formation of the TS. It is the case of the antibody 1F7 catalyzing the transformation of chorismate into prephenate," which stabilizes, thanks to several hydrogen bonds and an ionic bond between an arginine (Arg H95) and a carboxylate substituent of the substrate, the conformation of the chorismate which will give rise to the TS in a chair conformation for this reaction (Figure 11). 

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.

Fig. 5. Comparison of suppression efficiencies of five tRNAs A in T4 lysozyme at site 82, and B in chorismate mutase at site 88. Suppression efficiencies are defined as the amount of full-length protein divided by the sum of the full-length and truncated protein produced in each reaction. The suppression efficiencies shown represent the average of two trials. The tRNAs are identified below each bar Y yeast, E E. coli T Tetrahymena rt readthrough (un-acylated tRNA) V acylated with valine hE acylated with homoglutamate. Reprinted with permission [33]...

This enzyme [EC 4.1.3.27] catalyzes the reaction of chorismate with glutamine to generate anthranilate, pyruvate, and glutamate. In certain species, this enzyme is part of a multifunctional protein together with one or more other components of the system for the biosynthesis of tryptophan (Le., indole-3-glycerol-phosphate synthase, anthranilate phosphoribosyltransferase, tryptophan synthase, and phosphoribosylanthranilate isomerase). The anthranilate synthase that is present in these complexes has been reported to be able to utilize either glutamine or ammonia as the nitrogen source. However, it has also been reported that when anthranilate synthase is separated from this complex, only ammonia can serve as a substrate. 

Prephenate dehydrogenase [EC 1.3.1.12] catalyzes the reaction of prephenate with NAD+ to produce 4-hydro-xyphenylpyruvate, carbon dioxide, and NADH. This enzyme in enteric bacteria also possesses chorismate mutase activity and converts chorismate into prephenate. Prephenate dehydrogenase (NADP+) [EC 1.3.1.13] catalyzes the reaction of prephenate with NADP+ to produce 4-hydroxyphenylpyruvate, carbon dioxide, and NADPH. 

Inhibition of Chorismate Synthase Shikimic and quinic acids are used by microorganisms, fungi, and superior plants for the synthesis of essential aromatic amino acids from acyclic sugars. Fluorinated analogues of substrates and reaction intermediates have been synthesized in order to inhibit enzymes involved in... 

The hydrophobic effects between the apolar gronps involved in the Diels-Alder reaction also occnr when the apolar gronps belong to the same molecules, and thus should also be beneficial to the Claisen rearrangement. The nonenzymatic rearrangement of chorismate to prephenate occurs 100 times faster in water than in methanol (Copley and Knowles, 1987 Grieco et al., 1989). 

All carbons are derived from either erythrose 4-phosphate (light purple) or phosphoenolpyruvate (pink). Note that the NAD+ required as a cofactor in step (3) is released unchanged it may be transiently reduced to NADH during the reaction, with formation of an oxidized reaction intermediate. Step (6) is competitively inhibited by glyphosate (COO—CH2—NH—CH2—PO ), the active ingredient in the widely used herbicide Roundup. The herbicide is relatively nontoxic to mammals, which lack this biosynthetic pathway. The chemical names quinate, shikimate, and chorismate are derived from the names of plants in which these intermediates have been found to accumulate. 

In the tryptophan branch (Fig. 22-17), chorismate is converted to anthranilate in a reaction in which glutamine donates the nitrogen that will become part of the indole ring. Anthranilate then condenses with PRPP. The indole ring of tryptophan is derived from the ring carbons and amino group of anthranilate plus two carbons derived from PRPP. The final reaction in the sequence is catalyzed by tryptophan synthase. This enzyme has an a2/32 subunit structure and can be dissociated into two a subunits and a /32 subunit that catalyze different parts of the overall 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-... 

Elimination of P from 5-enolpyruvylshikimate 3-P (Eq. 25-3 and Fig. 25-1, step g) produces chorismate.30 The 24-kDa chorismate synthase, which catalyzes this reaction, requires for activity a reduced flavin. Although there is no obvious need for an oxidation reduction coenzyme, there is strong evidence that the flavin may play an essential role in catalysis, perhaps via a radical mechanism.31-331 ... 

Chemical properties appropriate to a compound found at a branch point of metabolism are displayed by chorismic acid. Simply warming the compound in acidic aqueous solution yields a mixture of prephen-ate and para-hydroxybenzoate (corresponding to reactions h and l of Fig. 25-1). Note that the latter reaction is a simple elimination of the enolate anion of pyruvate. As indicated in Fig. 25-1, these reactions correspond to only two of several metabolic reactions of the chorismate ion. In E. coli the formation of phe-nylpyruvate (steps h and i, Fig. 25-1) is catalyzed by a single protein molecule with two distinctly different enzymatic activities chorismate mutase and prephenate dehydratase.34-36 However, in some organisms the enzymes are separate.37 Both of the reactions catalyzed by these enzymes also occur spontaneously upon warming chorismic acid in acidic solution. The chorismate mutase reaction, which is unique in its mechanism,373 is discussed in Box 9-E. Stereochemical studies indicate that the formation of phenylpyruvate in Fig. 25-1, step z, occurs via a... 

Figure 25-2 The biosynthesis of tryptophan from chorismate and related synthetic reactions-...

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