EPSP Synthase by Glyphosate

On the basis of steady-state kinetics, glyphosate behaves as a reversible and competitive inhibitor of the forward reaction with respect to PEP and as an uncompetitive inhibitor with respect to shikimate 3-phosphate if the backward reaction is considered, then glyphosate is competitive with phosphate and uncompetitive with psp 14,16,18,22,23,25-31 kinetic patterns 

EPSP synthase appears to be unique in that, unlike all other PEP-utilizing enzymes studied, PEP is bound as the second substrate. This has a clear mechanistic link in that generally glyphosate does not inhibit enzymes utilizing PEP as a substrate. The only close analogy to the EPSP synthase-catalyzed reaction is found with UDP-N-acetylmuramic acid (UDP-NAM) synthase. Like EPSP synthase, this enzyme also transfers the enolpyruvyl 

Escherichia coli Escherichia coli Escherichia coli Klebsiella pneumoniae Klebsiella pneumoniae Neurospora crassa Candida maltosa Corydalis sempervirens Nicotiana silvestris Pisum sativum Sorghum bicolor 

UDP-NAM synthase and protect the enzyme against inactivation by cysteine-alkylating agents.  

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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.

Table 2,2. Structure-Function Relations of the Inhibition of arom EPSP Synthase by Glyphosate and Similar Compounds"...

The effective inhibition of EPSP synthase by glyphosate is essential evidence that this enzyme is the target site for herbicidal action. Even more... 

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. 

EPSP synthase catalyzes the synthesis of EPSP by an addition-elimination reaction through the tetrahedral intermediate shown in Fig. 2a. This enzyme is on the shikimate pathway for synthesis of aromatic amino acids and is the target for the important herbicide, glyphosate, which is the active ingredient in Roundup (The Scotts Company EEC, Marysville, OH). Transient-state kinetic studies led to proof of this reaction mechanism by the observation and isolation of the tetrahedral intermediate. Moreover, quantification of the rates of formation and decay of the tetrahedral intermediate established that it was tmly an intermediate species on the pathway between the substrates (S3P and PEP) and products (EPSP and Pi) of the reaction. The chemistry of this reaction is interesting in that the enzyme must first catalyze the formation of the intermediate and then catalyze its breakdown, apparently with different requirements for catalysis. Quantification of the rates of each step of this reaction in the forward and reverse directions has afforded a complete description of the free-energy profile for the reaction and allows... 

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. 

Fig. 4. Kinetics of glyphosale binding to EPSP synthase. (A) The change in protein fluorescence at i40 nm was measured by stopped-flow methods after mixing enzyme (4.5 fiM) with glyphosate (30 fiM) and S3P (250 /xM). The smooth line shows a fit to a single exponential with a rate of 4.5 sec. (B) The glyphosate concentration dependence of the observed rate was measured in the presence of a saturating concentration of S3P (250 fiM). (C) The S3P concentration dependence of the rate was measured in the presence of a fixed concentration of glyphosate (200 fiM). Reproduced with permission from (33).

The conversion of shikimate-3-phosphate (7) to 5-enolp-yruvylshikimate (EPSP) (8) by 5-enolpyruvylshikimate-3-phosphate synthase represents a rare type of reaction in which the enolpyruvate fragment of phosphoenolpyruvate (2) is transferred to a molecule of (— )-shikimate-3-phos-phate (7) (Figure 7.1). The herbicide glyphosate (10) blocks the enzyme that catalyzed this reaction (Fig. 7.3). In 1984, sales of this herbicide totalled 480 million (Amrhein, 1986 Floss, 1986). 

While the scope of our contribution does not permit a full discussion of the intricate question of the compart-mentation of the shikimate pathway in the cells of higher plants,(see also Chapter 3 by R.A. Jensen in this volume) the following observations are relevant to the mode of action of glyphosate using aqueous cell fractionation procedures, it has been shown that EPSP synthase in pea seedling shoots,sunflower cotyledons, and young spinach leaves (Joop and Amrhein, unpublished) is predominantly chloroplastic. Fractionation of freeze-stopped spinach leaves in nonaqueous media resulted in the recovery of >. 90% of EPSP synthase in the chloroplast fraction, and j<... 

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