Characterization of the double mutant, Val-177/Asn-322, of the lactose permease.
The double mutant, Val-177/Asn-322, was investigated with regard to its ability to transport H+ and galactosides. In downhill lactose transport assays, the wild-type strain had a Km value for lactose uptake of 0.9 mM and a Vmax of 0.65 mumol lactose/min.mg protein while the mutant had a significantly higher Km value of 1.9 mM but a similar Vmax of 0.49 mumol/min.mg protein. In spite of its moderate ability to transport lactose downhill, the Val-177/Asn-322 mutant exhibited the striking property of being completely defective in the uphill accumulation of lactose or methyl-beta-D-thiogalactopyranoside. Direct measurements of H+ transport, however, showed that the mutant's defect in active accumulation is not due to a defect in the ability to transport H+ with lactose or methyl-beta-D-thiogalactopyranoside. The Val-177/Asn-322 mutant strain had a H+:lactose stoichiometry of 0.84 which was similar to that measured in the wild-type strain (0.68). These results are discussed with regard to the role His-322 plays in H+ transport, active accumulation of sugars, and sugar recognition.
Brooker
R J
RJ
Department of Genetics & Cell Biology, University of Minnesota, St. Paul 55108.
D001216:Asparagine; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004338:Drug Combinations; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics*; D008756:Methylgalactosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010957:Plasmids; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism; D014633:Valine
D004269:DNA, Bacterial; D004338:Drug Combinations; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; C009468:thiomethylgalactoside; D001216:Asparagine; C021499:lactose permease; D014633:Valine; D007785:Lactose