The kinetic mechanism of galactoside/H+ cotransport in Escherichia coli.
To determine the kinetic mechanism of galactoside active transport by the lactose/H+ cotransporter of Escherichia coli, galactoside binding and transport are studied in the absence and presence of delta mu H+. For several reasons, the substrate beta-D-galactosyl-1-thi-beta-D-galactoside (GalSGal) is preferred over lactose. In the absence of delta mu H+, the cotransporter retains high affinity for GalSGal, and the affinity is the same on both sides of the membrane. At physiological pH, the cotransporter is protonated and the dissociation constant for H+ may be 50 pM. The cosubstrates bind in a random fashion. An isomerization of the cotransporter corresponding to reorientation of the binding sites is rate-determining. When delta mu H+ is imposed, two reorientations become faster, and one becomes slower. The affinity of the cotransporter for GalSGal on both sides of the membrane is unchanged. The inability of the cotransporter to bring the accumulation of galactoside into equilibrium with delta mu H+ at high galactoside concentrations can be explained without postulating uncoupled fluxes of galactoside or H+ across the membrane (leaks). The formation of the ternary carrier-H+-galactoside complex on the cytoplasmic side of the membrane with increasing internal levels of sugar and the rapidity of galactoside exchange inhibit net influx of galactoside and favor exchange. Net transport is slow at high galactoside levels. Thus, the cotransporter can self-regulate transport without uncoupling H+ and galactoside fluxes. Because the values of delta mu H+ during binding and transport studies were measured, these results can be subjected to a quantitative analysis.
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Wright
D001693:Biological Transport, Active; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D006096:Gramicidin / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism
D029968:Escherichia coli Proteins; D005697:Galactosides; D006027:Glycosides; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; D006096:Gramicidin; D006859:Hydrogen; C031803:thiodigalactoside; C021499:lactose permease