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Nonenzymatic Breakdown of the Tetrahedral (α-Carboxyketal Phosphate) Intermediates of MurA and AroA, Two Carboxyvinyl Transferases. Protonation of Different Functional Groups Controls the Rate and Fate of Breakdown

2003; American Chemical Society; Volume: 125; Issue: 41 Linguagem: Inglês

10.1021/ja0349655

1943-2984

Bartosz Byczynski, Shehadeh Mizyed, Paul J. Berti,

Metal-Catalyzed Oxygenation Mechanisms

The mechanisms of nonenzymatic breakdown of the tetrahedral intermediates (THIs) of the carboxyvinyl transferases MurA and AroA were examined in order to illuminate the interplay between the inherent reactivities of the THIs and the enzymatic strategies used to promote catalysis. THI degradation was through phosphate departure, with C−O bond cleavage. It was acid catalyzed and dependent on the protonation state of the carboxyl of the α-carboxyketal phosphate functionality, with ionizations at pKa = 3.2 ± 0.1 and 4.3 ± 0.1 for MurA and AroA THIs, respectively. The solvent deuterium kinetic isotope effect for MurA THI at pL 2.0 was 1.3 ± 0.4, consistent with general acid catalysis. The pKa's suggested intramolecular general acid catalysis through protonation of the bridging oxygen of the phosphate, though H3O+ catalysis was also possible. The product distribution varied with pH. The dominant breakdown products were {pyruvate + phosphate + R−OH} (R−OH = UDP-GlcNAc or shikimate 3-phosphate) at all pH's, particularly low pH. At higher pH's, increasing proportions of ketal, arising from intramolecular substitution of phosphate by the adjacent hydroxyl and the enolpyruvyl products of phosphate elimination were observed. With MurA THI, the product distribution fitted to pKa's 1.6 and 6.2, corresponding to the expected pKa's of a phosphate monoester. C−O bond cleavage was demonstrated by the lack of monomethyl [33P]phosphate formed upon degrading MurA [33P]THI in 50% methanol. General acid catalysis through the bridging oxygen is consistent with the location of the previously proposed general acid catalyst for THI breakdown in AroA, Lys22.