Inhibition EPSP synthase

ENZ enzyme assays, SC structural composition, MM molecular methods, IL isotopic labeling, IF isotopic fractionation, INH inhibition studies, UNK unknown, LOX lipoxogenase, EPSP synthase 5-enolpyruvylshikimate-3-phosphate, SDH shikimate dehydrogenase, PAL phenylalanine ammonium lyase, PKS polyketide synthase, NRPS nonribosomal peptide synthase 1 Gerwick 1999 2 Liu et al. 1994 3 Boonprab et al. 2003 4 Cvejic and Rohmer 1999 5 Disch et al. 1998 6 Chikaraishi et al. 2006 7 Schwender et al. 2001 8 Schwender et al. 1997 9 Mayes et al. 1993 10 Shick et al. 1999 11 Richards et al. 2006 12 Bouarab et al. 2004 13 Pelletreau et al., unpublished data 14 Dittman and Weigand 2006 15 Rein and Barrone 1999 Empty columns imply no direct evidence of these enzymes from these systems... 

Enolpyruvate shikimate-3-phosphate synthase (EPSPS) is the enzyme that catalyzes the condensation of shikimate-3-phosphate with phosphoenolpymvate. The corresponding difluorophosphonate (phosphoenolpymvate analogue) irreversibly inhibits EPSPS. The mechanism of the inhibition by difluorophosphonate is similar to that reported for MurZ inhibition (Figure 7.31). ... 

A related mechanism is utilized in the biosynthesis of UDP-muramic acid (Eq. 20-6) J There is an enolpyruvoyl adduct analogous to that of EPSP synthase a proposed enolpyruvoyl-enzyme adduct with Cys 115 is not on the major path.k/1 However, this enzyme is not inhibited by glyphosate.1... 

The condensation of phospho-shikimic acid with phosphoenol pyruvate is catalyzed by the enzyme 3-enoylpyruvoylhikimate-5-phosphate synthase, or EPSP synthase for short. This reaction is specifically inhibited by the herbicide glyphosate. 

Jaworski (4) reported that growth inhibition of both plant and microbes by glyphosate could be reversed by aromatic amino acids. Further work of Amrhein and his coworkers revealed that glyphosate inhibits the shikimate pathway enzyme, 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (5). This enzyme catalyzes the reaction shown in Figure 1. Glyphosate-treated plant and bacterial cultures accumulate shikimate and/or shikimate 3-phosphate (S3P), confirming that inhibition of EPSPS is at least a part of the in vivo mechanism of action of this herbicide (6, 7). 

It is clear from the above discussion ensure that glyphosate tolerance may be conferred to plants both by overproduction of wild type EPSPS as well as mutant EPSP synthases. It has been suggested that glyphosate may have multiple sites of action in plant cells (51-56). If this is true, mutant EPSPS enzymes would not confer glyphosate tolerance to plants, which is evidently not the case. It appears, therefore, that reports concerning the effect of glyphosate on other aspects of plant metabolism are due to secondary effects of the herbicide arising as a consequence of the inhibition of aromatic amino acid biosynthesis. 

The I50 (the glyphosate concentration necessary to inhibit 50% of the enzyme activity) of EPSP synthase was much higher in leaves of WR plants than in HR plants, in contrast to the situation inside the roots. 

For example, glyphosate inhibits the enzyme, EPSP (5-enolpyruvylshikimate 3-phosphate) synthase, that catalyzes a step in the synthesis of the aromatic amino acids. Similarly, both the imidazolinones and sulfonylureas inhibit acetolactate synthase (ALS), the enzyme that catalyzes the first step in the formation of branched-chain amino acids (11). Triazine herbicides act by binding to a specific protein in the thylakoid membranes of the chloroplasts, preventing the flow of electrons and inhibiting photosynthesis (12). 

C3b2. Inhibition of acetolactate and acetohydroxyacid synthases (B) C3b3. Inhibition of EPSP synthase (G) C3b4. Inhibition of glutamine synthetase (H)... 

In this chapter, the discussion will concentrate on two inhibitors with a reasonable claim to selective action on enz3ones related to the shikimate pathway glyphosate, which inhibits 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase and L-a-aminooxy-3 phenylpropionic acid (L-AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL) (Fig. 2). In addition to introducing a novel inhibitor of PAL, (R)-(l-amino-2-phenylethyl)phosphonic acid (APEP), previous and current efforts to design inhibitors of other shikimate pathway enzymes will be described. The treatment presented here will show that the deductions and predictions made on the basis of the abstract scheme in Figure 1 can be, and have been, tested on the basis of the real pathway presented in Figure 2. 

All published reports on the inhibition of EPSP synthase by glyphosate agree that the inhibition is competitive with respect to PEP. Table 1 surveys the published Ki values. The values must be compared with caution, however, because the conditions under which they were... 

Table 1. values for competitive inhibition, with respect to PEP, of EPSP synthases by glyphosate. 

Fig. 7. Lineweaver-Burk plots of inhibition of E. coli EPSP synthase by glyphosate with (a) PEP or (b) S-3-P as variable substrates. (c) Scatchard plot of binding of glyphosate to E. coli EPSP synthase in the absence of substrates.

Tissues and cultured cells accumulate predominantly shikimate, rather than S-3-P, in the presence of glyphosate, and in cultured buckwheat cells shikimate was shown to accumulate in the vacuole.As neither S-3-P nor shikimate compete with glyphosate for binding sites on EPSP synthase, and as PEP does not accumulate in glyphosate-treated cells (Amrhein, unpublished), the inhibition of EPSP synthase is not overcome. This example illustrates that in order to effectively inhibit an enzyme vivo the inhibitor need not necessarily be of the tight-binding or suicide (Kcat active-site-directed irreversible) type. 

TABLE 3. Inhibition of EPSP synthase by fluorine-containing analogs of the tetrahedral adduct. 

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