Changing the lactose permease of Escherichia coli into a galactose-specific symporter.
N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and beta,d-galactopyranosyl 1-thio-beta,D-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [(14)C]NEM. Remarkably, however, D-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consistently, competitive inhibition of [(14)C]galactose transport by lactose, melibiose, or beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected. The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected. In addition, although the conservative mutation Ala-122 --> Ser causes only mild inhibition of lactose transport, the mutations Ala-122 --> Phe and Ala-122 --> Tyr lead to marked inhibition. In contradistinction, none of these replacements has a marked effect on galactose transport. The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the non-galactosyl moiety of D-galactopyranosides. This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of D-galactopyranosides.
Guan
Lan
L
Howard Hughes Medical Institute, Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662.
Sahin-Toth
Miklos
M
Kaback
H Ronald
HR
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D005690:Galactose / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D013379:Substrate Specificity; D027981:Symporters / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D004187:Disaccharides; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D027981:Symporters; C023550:galactose-binding protein; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide; D000409:Alanine; D005690:Galactose