Phosphoryl exchange reaction catalyzed by enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Kinetic characterization.

Abstract

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 a- ketobutyrate 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.In their initial characterization of the bacterial phosphotransferase system, Kundig et al. (1) showed that the phosphorylation of sugars with phosphoenolpyruvate depended