Functional reconstitution of the purified phosphoenolpyruvate-dependent mannitol-specific transport system of Escherichia coli in phospholipid vesicles: coupling between transport and phosphorylation.
Purified mannitol-specific enzyme II (EII) from Escherichia coli was reconstituted into phospholipid vesicles with the aid of a detergent-dialysis procedure followed by a freeze-thaw sonication step. The orientation of EII in the proteoliposomes was random. The cytoplasmic moiety of the inverted EII could be removed with trypsin without effecting the integrity of the liposomal membrane. This enabled us to study the two different EII orientations independently. The population of inverted EII molecules was monitored by measuring active extrusion of mannitol after the addition of phosphoenolpyruvate, EI, and histidine-containing phosphocarrier protein (HPr) at the outside of the vesicles. The population of correctly oriented EII molecules was monitored by measuring active uptake of mannitol with internal phosphoenolpyruvate, EI, and HPr. A low rate of facilitated diffusion of mannitol via the unphosphorylated carrier could be measured. On the other hand, a high phosphorylation activity without translocation was observed at the outside of the liposomes. The kinetics of the phosphoenolpyruvate-dependent transport reaction and the nonvectorial phosphorylation reaction were compared. Transport of mannitol into the liposomes via the correctly oriented EII molecules occurred with a high affinity (Km, lower than 10 microM) and with a relatively low Vmax. Phosphorylation at the outside of the liposomes catalyzed by the inverted EII molecules occurred with a low affinity (Km of about 66 microM), while the maximal velocity was about 10 times faster than the transport reaction. The latter observation is kinetic proof for the lack of strict coupling between transport and phosphorylation in these enzymes.
Elferink
M G
MG
Department of Physical Chemistry, University of Groningen, The Netherlands.
Driessen
A J
AJ
Robillard
G T
GT
D001693:Biological Transport, Active; D002462:Cell Membrane / Q000648:ultrastructure; D002474:Cell-Free System; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism*; D008854:Microscopy, Electron; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011510:Proteolipids
D011510:Proteolipids; C020485:proteoliposomes; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System