Role of glutamate-126 and arginine-144 in the lactose permease of Escherichia coli.
Several previous studies have suggested that glutamate-126 and arginine-144 in the lactose permease of Escherichia coli form an ion pair that is essential for sugar binding. To further investigate the role of these residues, E126Q, R144Q, and R144S mutants were made. The R144Q and R144S strains, which had negligible levels of transport, were used as parental strains to isolate suppressor mutations that partially restored sugar transport. The R144Q parent only yielded first-site revertants, but the R144S strain produced three types of second-site replacements: E126Q, V229A, and L330R. In downhill transport assays, the E126Q strain was able to transport lactose at low levels, with an apparent K(m) 3-fold higher than the wild-type strain but a severely depressed apparent V(max). A triple mutant, E126Q/R144S/V229A, showed a relatively robust V(max) value for downhill transport and could actively accumulate lactose against a concentration gradient. Taken together, these results indicate that Glu-126 and Arg-144 are not essential for sugar binding. An alternative explanation for their role in maintaining secondary structure is discussed.
Johnson
Jerry L
JL
Department of Genetics, Cell Biology and Development and Biotechnology Institute, University of Minnesota, St. Paul, Minnesota 55108, USA.
Brooker
Robert J
RJ
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001692:Biological Transport / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D016153:Genes, Suppressor; D005947:Glucose / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary / Q000235:genetics; D027981:Symporters; D014170:Transformation, Genetic
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D018698:Glutamic Acid; C021499:lactose permease; D001120:Arginine; D005947:Glucose; D007785:Lactose; D005690:Galactose