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Proton transfer to carbon atoms is a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction. We performed transition state analysis using kinetic isotope effects (KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a methylene carbon (C3) protonation, as an analog of AroA's reverse reaction. As part of this analysis, an inorganic phosphate scavenging system was developed to remove phosphate which, though present in microscopic amounts in solution, is ubiquitous. The reaction was stepwise, with irreversible C3 protonation to form an EPSP cation intermediate; that is, an AH(‡)*AN mechanism. The large experimental 3-(14)C KIE, 1.032 ± 0.005, indicated strong coupling of C3 with the motion of the transferring proton. Calculated 3-(14)C KIEs for computational transition state models revealed that the transition state occurs early during C3-H(+) bond formation, with a C3-H(+) bond order of ≈0.24. The observed solvent deuterium KIE, 0.97 ± 0.04, was the lowest observed to date for this type of reaction, but consistent with a very early transition state. The large 2-(14)C KIE reflected an "electrostatic sandwich" formed by Asp313 and Glu341 to stabilize the positive charge at C2. In shifting the transition state earlier than the acid-catalyzed reaction, AroA effected a large Hammond shift, indicating that a significant part of AroA's catalytic strategy is to stabilize the positive charge in the EPSP cation. A computational model containing all the charged amino acid residues in the AroA active site close to the reactive center showed a similar Hammond shift relative to the small transition state models.

Enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) catalyzes the sixth step in aromatic amino acid biosynthesis. It forms EPSP from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) in an addition/elimination reaction that proceeds through a tetrahedral intermediate. In spite of numerous mechanistic studies, the catalytic roles of specific amino acid residues remain an open question. Recent experimental evidence for cationic intermediates or cationic transition states, and a consideration of the catalytic imperative, have guided this study on the catalytic roles of Lys22 (K22), Asp313 (D313), and Glu341 (E341). Steady-state and pre-steady-state kinetics and protein stability studies showed that mutations of D313 and E341 caused k(cat) to decrease up to 30,000-fold and 76,000-fold, respectively, while the effects on K(M) were modest, never more than 40-fold. Thus, both are identified as catalytic residues. In an active site that is overwhelmingly positively charged, the D313 and E341 side chains are positioned to form an "electrostatic sandwich" around the positive charge at C2 in cationic intermediates/transition states, stabilizing them and thereby promoting catalysis. Mutation of K22 showed large effects on K(M,S3P) (100-fold), K(M,PEP) (>760-fold), and up to 120-fold on k(cat). Thus, K22 had roles in both substrate-binding and transition-state stabilization. These results support the identification of E341 and K22 as general acid/base catalytic residues.

Enolpyruvylshikimate-3-phosphate synthase (AroA, also called EPSP synthase) is a carboxyvinyl transferase involved in aromatic amino acid biosynthesis, forming EPSP from shikimate 3-phosphate and phosphoenolpyruvate. Upon extended incubation, EPSP ketal, a side product, forms by intramolecular nucleophilic addition of O4 to C2' of the enolpyruvyl group. The catalytic significance of this reaction was unclear, as it was initially proposed to arise from nonenzymatic breakdown of tetrahedral intermediate that had dissociated from AroA. This study shows that EPSP ketal formed in AroA's active site, not nonenzymatically, by demonstrating its formation in the presence of excess AroA. It formed both in the normal reaction and during AroA-catalyzed EPSP hydrolysis. In addition, nonenzymatic EPSP hydrolysis was studied to elucidate the catalytic imperative for enolpyruvyl reactions. Hydrolysis was acid-catalyzed, with a rate enhancement of >5 x 10(8)-fold. There was no detectable EPSP breakdown after 16 days at 90 degrees C in 1 M KOH, a solution that is 1000-fold more nucleophilic than neutral aqueous solutions. Thus, an unactivated enolpyruvyl group is not susceptible to nucleophilic attack. Enzymatic EPSP ketal formation therefore requires enolpyruvyl activation through protonation of C3' to form either a cationic intermediate or a highly cation-like transition state. Forming an EPSP cation requires the investment of considerable catalytic power by AroA. Such an intermediate is a potential target motif for inhibitor design.