Phosphoryl Exchange Reaction Catalyzed by Enzyme I of the Bacterial Phosphoeno 1 pyruvate : Sugar Phosphotransferase System

Enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system catalyzes phosphoryl transfer from phosphoenolpyruvate to the heat-stable phosphoryl carrier protein, HPr. Enzyme I also catalyzes a rapid phosphoryl exchange reaction in which the phosphoryl moiety of phosphoenolpyruvate is transferred to pyruvate. Additionally, Enzyme I plus HPr catalyze the slow hydrolysis of phosphoenolpyruvate to pyruvate and inorganic phosphate. A simple and quantitative assay was developed for measuring these reactions. “C-Labeled phosphoenolpyruvate or pyruvate was used, and the formation of [‘4C]pyruvate or phosphoenolpyruvate was measured, respectively. The two radioactive products were separated by conversion of pyruvate to the dinitrophenylhydrazone derivative and extraction into ethyl acetate. Employing this assay, the Enzyme I-catalyzed phosphoryl exchange and sugar phosphorylation reactions showed sigmoidal kinetics when reaction rate was plotted versus Enzyme I concentration. The enzyme was activated by M g + , Mn2+, and Co2+ but not by other divalent cations tested. The pH optimum in the presence of Mg2’ was 1.5. Kinetic binding constants were estimated as follows: P-enolpyruvate, 0.4 m ~ ; pyruvate, 2 m ~ ; Mg“’, 2 mm; P-enolpyruvate (bound to phospho-Enzyme I), 2.5 m ~ ; and pyruvate (bound to free Enzyme I), 20 m ~ . Low concentrations of bromopyruvate irreversibly inactivated the enzyme, while oxalate appeared to be a potent transition state inhibitor. Enzyme I catalyzed exchange reactions when aketobutyrate or fl-hydroxypyruvate replaced pyruvate, but higher homologues of pyruvate were essentially inactive. Bacterial extracts catalyzed the hydrolysis of phosphoenolpyruvate in the presence of low concentrations of Co2+. This activity was not attributable to a known enzyme constituent of the PTS.