EPSP synthase, enzyme intermediates

Anderson KS, Sammons RD, Leo GC, Sikorski JA, Benesi AJ, Johnson KA. Observation by 13C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site. Biochemistry 1990 29 1460-1465. 

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.

Figure 2b shows the results of a rapid-quench single-turnover experiment performed with EPSP synthase with enzyme in excess over the radiolabeled substrate, PEP. The data show the transient formation and decay of the tetrahedral intermediate, which led to its subsequent isolation and stmcture determination. 

Anderson KS, Johnson KA. Kinetic and Structural Analysis of Enzyme Intermediates Lessons from EPSP Synthase. Chem. Rev. 1990 90 1131-1149. 

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). 

EPSP synthase A tetrahedral ketal phosphate enzyme intermediate 672... 

Much earlier studies on EPSP synthase with radiolabeled substrates suggested the existence of an enzyme intermediate. ° ° There were two plausible mechanisms as illustrated in Scheme 4 (1) a tetrahedral intermediate formed by protonation of the C-3 carbon of PEP, and subsequent attack of the 5 -OH of shikimate-3-phosphate (S3P) to form an intermediate that was not covalently bound to the enzyme as illustrated in the scheme (pathway a), and a covalent intermediate involving a protonated form of PEP that becomes covalently attached to the enzyme through an active site nucleophile during catalysis (pathway b). 

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. ... 

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 formation of a covalent phospholactoyl-enzyme adduct is a major distinction between the mechanisms of catalysis by UDP-GlcNAc enolpyruvoyl transferase (MurZ) and EPSP synthase. Nonetheless, the structural and functional homologies suggest at least some common mechanistic features, as indicated by the isolation of similar tetrahedral intermediates in both enzyme reactions. 

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. 

A number of analogues that mimic the intermediate formed during the EPSP nthase conversion of shikimic-3-phosphate into EPSP (5-enolpyruvyl shikimate-3-phosphate) have been prepared and tested as inhibitors of the enzyme. The best was shown to be 63. (Z)-3-Fluoro-phosphoenolpyruvate functions as a pseudosubstrate for EPSP synthase producing in one step the same adduct 63, but as an isomeric mixture. ... 

Figure 1. Hypothetical mechanism for shuttling of intermediates of the common aromatic pathway between plastidic and cytosolic compartments. Enzymes denoted with an asterisk (DAHP synthase-Co, chorismate mutase-2, and cytosolic anthranilate synthase) have been demonstrated to be isozymes located in the cytosol. DAHP molecules from the cytosol are shown to be shuttled into the plastid compartment in exchange for EPSP molecules synthesized within the plastid. Abbreviations C3, phosphoenolpyruvate C4, erythrose 4-P DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate EPSP, 5-enolpyruvylshikimate 3-phosphate CHA, chorismate ANT, anthranilate TRP, L-tryptophan PPA, prephenate AGN, L-arogenate TYR, L-tyrosine and PHE, L-phenylalanine.

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