Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system

Abstract

The inducible, mannitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system has been purified approximately 230-fold from Escherichia coli membranes. The enzyme, initially solubilized with deoxycholate, was first subjected to hydrophobic chromatography on hexyl agarose and then purified by several ion exchange steps in the presence of the nonionic detergent, Lubrol PX. The purified protein appears homogeneous by several criteria and probably consists of a single kind of polypeptide chain with a molecular weight of 60,000 (+/- 5%). In addition to catalyzing phosphoenolpyruvate-dependent phosphorylation of mannitol in the presence of the soluble enzymes of the phosphotransferase system, the purified Enzyme II also catalyzes mannitol 1-phosphate:mannitol transphosphorylation in the absence of these components. A number of other physical and catalytic properties of the enzyme are described. The availability of a stable, homogeneous Enzyme II should be invaluable for studying the mechanism of sugar translocation and phosphorylation catalyzed by the bacterial phosphotransferase system.