The substrate-binding site in the lactose permease of Escherichia coli.
Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed fluorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 --> Cys was also studied. In marked contrast to Val-331 --> Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 --> Ala/Val-331 --> Cys, Arg-144 --> Ala/Val-331 --> Cys, or Arg-144 --> Lys/Val-331 --> Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 --> Ala or Arg-144 --> Ala and, to a lesser extent, Arg-144 --> Lys cause a red-shift in the emission spectrum and render the fluorophore more accessible to I-. The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H+ translocation pathway at the interface between helices IX and X.
Venkatesan
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Howard Hughes Medical Institute, Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662, USA.
Kaback
H R
HR
D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents; D027981:Symporters
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C021499:lactose permease; D005033:Ethylmaleimide