Mannitol-specific enzyme II of the bacterial phosphotransferase system. I. Properties of the purified permease.
The integral membrane protein responsible for the transport and phosphorylation of D-mannitol in Escherichia coli, the mannitol-specific Enzyme II of the phosphotransferase system (Mr = 60,000), has been purified to apparent homogeneity using a modification of a previously published procedure (Jacobson, G. R., Lee, C. A., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252). The purified enzyme was dependent on Lubrol PX and phospholipid for maximal activity. It catalyzed both the phosphoenolpyruvate- and the mannitol 1-phosphate-dependent phosphorylation of D-mannitol with high specificity for the accepting sugar and the phosphoryl donor. Both mannitol and mannitol 1-phosphate gave strong substrate inhibition at neutral pH in the transphosphorylation reaction catalyzed by the purified mannitol Enzyme II, while no substrate inhibition by mannitol was observed for the phosphoenolpyruvate-dependent reaction. The purified enzyme did not catalyze hydrolysis of mannitol 1-phosphate, a product of both reactions. Antibody directed against the mannitol Enzyme II inhibited the phosphoenolpyruvate-dependent activity to a greater extent than the transphosphorylation activity. Limited proteolysis with trypsin rapidly inactivated both purified and membrane-bound mannitol Enzyme II, and the purified protein was concomitantly cleaved into fragments with apparent molecular weights of about 29,000. These results show that although the mannitol Enzyme II is an integral membrane protein, a considerable portion of its polypeptide chain must also extend into a hydrophilic environment, presumably the cytoplasm.
D003429:Cross Reactions; D029968:Escherichia coli Proteins; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000276:immunology / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology; D012486:Salmonella typhimurium / Q000201:enzymology; D013211:Staphylococcus aureus / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010743:Phospholipids; D000077423:Polidocanol; D011092:Polyethylene Glycols; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D014357:Trypsin