title: Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli.
abstract: Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.
authors.affiliation: Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles 90095-1662, USA.
authors.forename: M
authors.initials: M
authors.lastname: Sahin-Tóth
authors.forename: J
authors.initials: J
authors.lastname: le Coutre
authors.forename: D
authors.initials: D
authors.lastname: Kharabi
authors.forename: G
authors.initials: G
authors.lastname: le Maire
authors.forename: J C
authors.initials: JC
authors.lastname: Lee
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001693:Biological Transport, Active; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009598:Nitrophenylgalactosides / Q000737:chemistry; D011522:Protons; D013379:Substrate Specificity / Q000235:genetics; D027981:Symporters; D013862:Thiogalactosides / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D013862:Thiogalactosides; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D018698:Glutamic Acid; C031803:thiodigalactoside; C021499:lactose permease; D001120:Arginine; D007785:Lactose; D005033:Ethylmaleimide