EPSPS-catalyzed reaction

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... 

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... 

Figure 2 Intermediate in the EPSP synthase pathway, (a) The mechanism of the reaction catalyzed by EPSP synthase is shown. The reaction proceeds by an addition-elimination mechanism via a stable tetrahedral intermediate, (b) A single turnover reaction is shown in which 10- xM enzyme was mixed with 1 OO-m-M S3P and 3.5-riM radiolabeled PEP. Analysis by rapid-quench kinetic methods showed the reaction of PEP to form the intermediate, which then decayed to form EPSP in a single turnover. The smooth lines were computed from a complete model by numerical integration of the equations based on a global fit to all available data. Reproduced with permission from Reference 7.

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). 

EPSPS has been isolated from both microorganisms and plants, and several of its properties have been studied. The bacterial and plant enzymes are mono functional with molecular mass of 44-48 kD (8-15). The fungal enzyme is a part of the multifunctional arom complex which catalyzes four other reactions of the shikimate pathway (16). While the bacterial enzymes show differences with respect to glyphosate sensitivity, the plant enzymes exhibit a much more narrow range of sensitivity (17). This accounts for the susceptibility of most plant species to glyphosate. 

The kinetics of formation and decay of a tetrahedral intermediate at the active site of EPSP synthase has been examined in both the forward and reverse directions, as shown in Fig. 9. The reaction catalyzed by EPSP synthase is an ordered, Bi Bi mechanism (Scheme XIX). 

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). 

EPSP synthase catalyzes a rare type of reaction in which the intact enolpyruvyl (carboxyvinyl) group is transferred from phosphoenolpyruvate (PEP) to the 5-hydroxyl group of shikimate 3-phosphate (Fig. 6). This type of reaction is encountered in only one other known enz3rmatic process the transfer of the enolpyruvyl group to UDP-N-... 

After silylation of epoxy lactone 1 and opening to the epoxy ester 18, we expected that application of Ganem s procedure for the introduction of the enolpyruvyl side chain would afford an intermediate appropriate for synthesis of EPSP. Unfortunately, the epoxide functionality is incompatible with the rhodium-catalyzed diazomalonate insertion reaction and the desired intermediate 19 is not obtained. The major product appears to result firom deoxygenation of the epoxide. 

Figure 7. Mechanism of the reaction catalyzed by EPSP synthase.

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