Mechanics of solute translocation catalyzed by enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.
The kinetics of binding of mannitol to enzyme IImtl embedded in the membrane of vesicles with an inside-out or a right-side-out orientation were analyzed at 4 degrees C in the absence of the phosphoryl group donor, P-HPr. The binding to the right-side-out oriented vesicles equilibrated too fast to be monitored by the flow dialysis technique. On the other hand, with the inside-out oriented membrane vesicles two conformational changes of the enzyme could be detected kinetically. One change involved a recruitment of binding sites from a state of the enzyme where the binding sites were inaccessible from the cytoplasmic volume. The second change involved a conformational change of the enzyme that followed upon the initial binding to the cytoplasmic-facing binding site leading to a state with a higher affinity for mannitol. Equilibrium binding to the inside-out and right-side-out oriented membrane vesicles at 4 degrees C indicated that the two transitions did not represent the translocation of the binding site, free and with mannitol bound to it, to the other side of the membrane. Instead, a model is proposed in which the conformational changes represent transitions from states with the binding pocket opened to the cytoplasmic side of the membrane to occluded states of the enzyme in which the binding sites, with or without mannitol bound, are not accessible to either side of the membrane.
Lolkema
J S
JS
Department of Biochemistry, University of Groningen, The Netherlands.
Dijkstra
D S
DS
Robillard
G T
GT
D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D004058:Diffusion; D004926:Escherichia coli / Q000201:enzymology*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008353:Mannitol / Q000378:metabolism*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
D046911:Macromolecular Substances; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System