Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions
✍ Scribed by Sanjay S. Khandekar; Gary R. Jacobson
- Publisher
- John Wiley and Sons
- Year
- 1989
- Tongue
- English
- Weight
- 575 KB
- Volume
- 39
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
Column chromatography of the Escherichiu coli mannitol permease (mannitolspecific enzyme II of the phosphotransferase system) in the presence of deoxycholate has revealed that the active permease can exist in at least two association states with apparent molecular weights consistent with a monomer and a dimer. The monomeric conformation is favored by the presence of mannitol and by the phosphoenolpyruvate (PEP)-dependent phosphorylation of the protein. The dimer is stabilized by inorganic phosphate (Pi), which also stimulates phospho-exchange between mannitol and mannitol 1-phosphate (a partial reaction in the overall PEPdependent phosphorylation of mannitol). Kinetic analysis of the phospho-exchange reaction revealed that Pi stimulates phospho-exchange by increasing the V , , of the reaction. A kinetic model for mannitol permease function is presented involving both conformations of the permease. The monomer (or a less-stable conformation of the dimer) is hypothesized to be involved in the initial mannitol-binding and PEP-dependent phosphorylation steps, while the stably associated dimer is suggested to participate in later steps involving direct phosphotransfer between the permease, mannitol and mannitol 1-phosphate.
Key words. sugar transport, bacterial phosphotransferase system, protein conformation, monomerh e r equihbruim
Escherichiu coli transports D-mannitol and a number of other sugars through its cytoplasmic membrane via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) first identified by Kundig et al. [l]. The PTS carries out the concomitant transport and phosphorylation of these sugars resulting in sugar phos-Abbreviations used: DTT, dithiothreitol; EI, enzyme I of PTS; HPr, heat-stable phosphocamer protein of the PTS; PAGE, polyacrylamide gel electrophoresis; PEP, phosphoenolpyruvate; Pi, inorganic phosphate; PMSF, phenylmethylsulfonylfluoride; PTS, phosphoenolpyruvate-dependent sugar phosphotransferase system; SDS, sodiumdodecylsulfate.