A Comparative Study of the Enzymatic and Non-Enzymatic Breakdowns of the Tetrahedral Intermediate of the MurA Reaction

Abstract

The mechanism of the non-enzymatic breakdown of the tetrahedral intermediate of MurA (a carboxyvinyl transferase) was determined in order to better understand the corresponding enzyme catalyzed reaction. The breakdown was general acid-catalyzed, with simultaneous C-O bond cleavage and protonation at the bridging oxygen of the phosphate leaving group. The carboxyl was shown to play an important role in the breakdown, with its pKₐ of 3.2 appearing in the rate 𝘷𝘦𝘳𝘴𝘶𝘴 pH profile. The product distribution varied with pH. At pH 1, the products were predominantly UDP-GIcNAc, pyruvate and phosphate. Increasing proportions of MurA ketal arising from intramolecular attack of the 4'-0H, and EP-UDP-GicNAc resulting from elimination were observed with higher pHs. The MurA ketal was structurally characterized, and shown to be analogous to the ketal formed by AroA, a related enzyme. pKₐs corresponding to those expected of phosphate monoesters (1.6 and 6.2) were found in the product distribution 𝘷𝘦𝘳𝘴𝘶𝘴 pH profile. Observation of these pKₐs supported the initial product of phosphate departure being an oxocarbenium ion-phosphate tight ion pair complex. A corresponding oxocarbenium ion intermediate in the enzymatic reaction could not be demonstrated.