Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli
| Autor: | Jaap Broos, Gertjan Veldhuis, Bert Poolman, Ruud M. Scheek |
|---|---|
| Přispěvatelé: | X-ray Crystallography, Enzymology, Molecular Dynamics, Faculty of Science and Engineering, Groningen Biomolecular Sciences and Biotechnology |
| Jazyk: | angličtina |
| Rok vydání: | 2005 |
| Předmět: |
PERMEASE
Time Factors RADIOCHEMICAL IMPURITIES Monosaccharide Transport Proteins Ultraviolet Rays Detergents Biophysics Biophysical Theory and Modeling Plasma protein binding Ligands LIGAND INTERACTIONS Absorption Polyethylene Glycols Phosphoenolpyruvate Escherichia coli medicine Computer Simulation Mannitol ENZYME-IIMTL Binding site Phosphoenolpyruvate Sugar Phosphotransferase System PHOSPHORYLATION Bradford protein assay Binding Sites COMPLEX Dose-Response Relationship Drug L-Lactate Dehydrogenase Chemistry Permease Escherichia coli Proteins Genetic Complementation Test Thrombin Membrane Transport Proteins Proteins Substrate (chemistry) Biological Transport Ligand (biochemistry) Dissociation constant Kinetics EQUILIBRIUM Biochemistry DEPENDENT PHOSPHOTRANSFERASE SYSTEM OLIGOPEPTIDE-BINDING-PROTEIN MEMBRANE Plasmids Protein Binding medicine.drug |
| Zdroj: | Biophysical Journal, 89(1), 201-210. CELL PRESS |
| ISSN: | 1542-0086 |
| DOI: | 10.1529/biophysj.105.062877 |
| Popis: | 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 KD-values in the nano- and micromolar range, respectively. However, reported KD-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 KD-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. |
| Databáze: | OpenAIRE |
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