EPSP synthase, enzyme intermediates reaction pathway

A series of rapid chemical quench experiments under single enzyme turnover conditions using radiolabeled S3P or PEP revealed that the tetrahedral ketal phosphate enzyme intermediate was formed as a new peak upon HPLC analysis with detection of the radiolabel. The time course revealed that the formation of the tetrahedral intermediate species paralleled the disappearance of PEP substrate and formation of the EPSP product thus establishing that it was a kinetically competent species. Isolation of the tetrahedral ketal phosphate intermediate using C-2 PEP and S3P as substrates coupled with rapid chemical quench was carried out in conjunction with H-, C-, and P- NMR to provide a definitive structure proof Thus with these studies we have satisfied the criteria for a true reaction intermediate in terms of a chemically plausible mechanism, structure proof, and kinetic competence. Additional studies support the mechanism for EPSP synthase described (Scheme 4, pathway a) including observation of the intermediate bound to the enzyme at internal equilibrium using solution NMR and C-2 PEP as well as using rapid freeze-quench/solid-state NMR studies. ... 

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.

Fio. 9. EPSP synthase single-turnover kinetics and EPSP synthase reaction pathway. (A) The disappearance and formation of PEP ( ), EPSP ( ), and intermediate (A) were monitored in the reverse direction. The reaction was initiated by mixing enzyme (10 /xM) and S3P (100 fiM) with radiolabeled PEP (3.5 /xAf). (B) The disappearance and formation of EPSP ( ), PEP ( ), and intermediate (A) were monitored in the reverse direction. The reaction was initiated by mixing enzyme (10 iiM) with phosphate (7.5 /zM) and radiolabeled EPSP (2.1 fiM). The curves were calculated by computer simulation using the fill kinetic pathway shown in Scheme XIX and the 12 individual rate constants (3). Reproduced with permission from (3). 

In summary, through the use of rapid chemical quench techniques, multiple studies demonstrated the formation of a single tetrahedral intermediate in the reaction pathway of EPSP synthase (Scheme 4, pathway a) which is formed by an attack of the 5-OH group of shikimate-3-phosphate on C-2 of PEP. A complete kinetic and thermodynamic description of this enzyme reaction pathway could be demonstrated, including the isolation and structural elucidation of a tetrahedral enzyme intermediate as originally proposed by Sprinson. This work established the catalytic mechanism and definitively showed that no covalent enzyme—PEP adduct is formed on the reaction pathway. Subsequent work using rapid mixing pulsed-flow ESI—MS studies and solution phase NMR " provides additional support for the catalytic pathway in Scheme 4, pathway a. 

The proposed cyclic ketal phosphate intermediate 2 in Scheme 6 has very similar functionality to the tetrahedral enzyme intermediates isolated and characterized in the EPSP synthase and UDP-GlcNAc enol-pyruvoyl transferase reactions. Although the tetrahedral ketal phosphate intermediate for EPSP synthase is quite labile at neutral or acidic pH, it is surprisingly stable at basic pH>12 (q/2=48h). Based upon these results, we might predict that intermediate 2 should be detectable by rapid chemical quench techniques if isolated under basic conditions even if it was formed only transiently at the enzyme active site. Although the mechanistic data described above suggest the catalytic pathway outlined in Scheme 6, there was no direct information in support of either intermediate. 

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