Mechanistic coupling of transport and phosphorylation activity by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.
Mannitol bound to enzyme IImtl could be trapped specifically by rapid phosphorylation with P-HPr. The assay was used to demonstrate transport of mannitol across the cytoplasmic membrane with and without phosphorylation of mannitol. The latter was 2-3 orders of magnitude slower. The fraction of bound mannitol molecules that was actually phosphorylated, the efficiency of the trap, was less than 50%. The efficiency was not very different for enzyme IImtl embedded in the membrane of vesicles with an inside-out orientation or solubilized in detergent. Subsequently, it is argued that the fraction of the bound mannitol molecules that was not phosphorylated dissociated into the cytoplasmic space. A model for the catalytic mechanism of enzyme IImtl is proposed on the basis of interpretations of the present experiments. The main features of the model are the following: (i) mechanistically, the coupling between transport and phosphorylation is less than 50%; (ii) in the physiological steady state of mannitol transport and metabolism, the coupling is 100%; (iii) phosphorylated enzyme IImtl catalyzes facilitated diffusion at a high rate; (iv) the state of phosphorylation of the cytoplasmic domain modulates the activity of the translocator domain; (v) the enzyme catalyzes phosphorylation of free cytoplasmic mannitol at least as fast as it catalyzes transport plus phosphorylation of free periplasmic mannitol.
Lolkema
J S
JS
Department of Physical Chemistry, University of Groningen, The Netherlands.
ten Hoeve-Duurkens
R H
RH
Dijkstra
D S
DS
Robillard
G T
GT
D001665:Binding Sites; D001693:Biological Transport, Active; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008353:Mannitol / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli