Enolpyruvyl Activation by Enolpyruvylshikimate-3-phosphate Synthase †

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.