Site-directed mutagenesis of lysine 319 in the lactose permease of Escherichia coli.
Lys319, which is on the same face of putative helix X as His322 and Glu325 in the lactose permease of Escherichia coli, has been replaced with Leu by oligonucleotide-directed, site-specific mutagenesis. Although previous experiments suggested that the mutation does not alter permease activity, we report here that K319L permease is unable to catalyze active lactose accumulation or lactose efflux down a concentration gradient. The mutant does catalyze facilitated influx down a concentration gradient at a significant rate; however, the reaction occurs without concomitant H+ translocation. The mutant also catalyzes equilibrium exchange at about 50% of the wild-type rate, but it exhibits poor counterflow activity. Finally, flow dialysis and photoaffinity labeling experiments with p-nitrophenyl alpha-D-galactopyranoside indicate that K319L permease probably has a markedly decreased affinity for substrate. The alterations described are not due to diminished levels of the mutated protein in the membrane, since immunological studies reveal comparable amounts of permease in wild-type and K319L membranes. It is proposed that Lys319, like Arg302, His322, and Glu325, plays an important role in active lactose transport, as well as substrate recognition.
Persson
B
B
Howard Hughes Medical Institute, Department of Physiology and Microbiology, University of California, Los Angeles 90024.
Roepe
P D
PD
Patel
L
L
Lee
J
J
Kaback
H R
HR
D000345:Affinity Labels; D001693:Biological Transport, Active / Q000187:drug effects; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D013329:Structure-Activity Relationship; D027981:Symporters
D000345:Affinity Labels; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C021499:lactose permease; D007785:Lactose