PMID:12011425
| Citation |
Guan, L, Sahin-Toth, M and Kaback, HR (2002) Changing the lactose permease of Escherichia coli into a galactose-specific symporter. Proc. Natl. Acad. Sci. U.S.A. 99:6613-8 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC124451 Online version:10.1073/pnas.102178299 |
| Keywords |
Alanine/genetics; Alanine/metabolism; Calcium-Binding Proteins; Cysteine/genetics; Cysteine/metabolism; Disaccharides/metabolism; Enzyme Inhibitors/pharmacology; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Ethylmaleimide/pharmacology; Galactose/metabolism; Lactose/metabolism; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Monosaccharide Transport Proteins/metabolism; Mutagenesis, Site-Directed; Periplasmic Binding Proteins; Substrate Specificity; Symporters/metabolism |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.