Subunit interactions of the Escherichia coli mannitol permease: correlation with enzymic activities.
A fraction of the phosphorylated form of the Escherichia coli mannitol permease (enzyme IIMtl) of the sugar phosphotransferase system can be extracted from the membrane in a dimeric form [Roossien, F.F., & Robillard, G.T. (1984) Biochemistry 23, 5682-5685]. Using E. coli minicells in which this protein can be specifically labeled with [35S]methionine, we show in this paper that part of the unphosphorylated form of enzyme IIMtl can also be extracted from the membrane as a dimer. We further demonstrate that both phosphoenolpyruvate-dependent phosphorylation of the permease and conditions promoting turnover of the enzyme decrease the amount of extractable dimer. Thus, the dimer of these forms of the enzyme appears to be less stable than that of the unmodified form, at least in detergent solution. In contrast, inorganic phosphate, which activates the permease-catalyzed phospho exchange between mannitol 1-phosphate and mannitol ("transphosphorylation"), stabilizes the dimer. These results support the hypothesis that the mannitol permease dimer is more active in transphosphorylation than the monomer. Treatment of minicell membranes with oxidizing agents produced heat-stable, high molecular weight aggregates of the permease on dodecyl sulfate gels, but no heat-stable dimer could be detected. The nonionic detergent Lubrol PX decreased the amount of dimer extractable at 30 degrees C with a concomitant increase in the monomeric form. These results suggest that the dimer depends predominantly on hydrophobic interactions for its stability and is not covalently cross-linked in that form by oxidizing agents.
D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D010710:Phosphates / Q000494:pharmacology; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; D010710:Phosphates; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli