Stoichiometry and substrate affinity of the mannitol transporter, EnzymeIImtl, from Escherichia coli.
Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeIImtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a low-affinity binding site with K(D)-values in the nano- and micromolar range, respectively. However, reported K(D)-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the discrepancies reported in literature, regarding both the variability in K(D)-values and the binding stoichiometry. By comparing the binding capacity obtained with flow dialysis with different methods to determine the protein concentration (UV-protein absorption, Bradford protein detection, and a LDH-linked protein assay to quantify the number of phosphorylation sites), we proved the existence of only one mannitol binding site per dimeric species of unphosphorylated EnzymeIImtl. Furthermore, the affinity of EnzymeIImtl for mannitol appeared to be dependent on the protein concentration and seemed to reflect the presence of an endogenous ligand. The dependency could be simulated assuming that >50% of the binding sites were occupied with a ligand that shows an affinity for EnzymeIImtl in the same range as mannitol.
Veldhuis
Gertjan
G
Department of Biochemistry and Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands.
Broos
Jaap
J
Poolman
Bert
B
Scheek
Ruud M
RM
D000042:Absorption; D001665:Binding Sites; D001692:Biological Transport; D003198:Computer Simulation; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D008024:Ligands; D008353:Mannitol / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry; D009004:Monosaccharide Transport Proteins; D010728:Phosphoenolpyruvate / Q000737:chemistry; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011092:Polyethylene Glycols; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors; D014466:Ultraviolet Rays
D003902:Detergents; D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011506:Proteins; D008353:Mannitol; D011092:Polyethylene Glycols; D010728:Phosphoenolpyruvate; D007770:L-Lactate Dehydrogenase; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D013917:Thrombin