PMID:12471022
| Citation |
Guan, L, Sahin-Tóth, M, Kálai, T, Hideg, K and Kaback, HR (2003) Probing the mechanism of a membrane transport protein with affinity inactivators. J. Biol. Chem. 278:10641-8 |
|---|---|
| Abstract |
Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala(122)-->Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (Deltamu(H(+))) markedly increases the rate of reaction between Cys(122) and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala(122)-->Cys mutation is combined with a mutation (Cys(154)-->Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and Deltamu(H(+)) has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala(122) in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism. Thus, these inactivators behave as unique suicide substrates. |
| Links |
PubMed Online version:10.1074/jbc.M211355200 |
| Keywords |
Affinity Labels; Catalysis; Cysteine; Escherichia coli; Escherichia coli Proteins; Galactosides/chemistry; Galactosides/metabolism; Glucosides/chemistry; Glucosides/metabolism; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/metabolism; Mesylates/chemistry; Mesylates/metabolism; Monosaccharide Transport Proteins; Protein Transport; Symporters |
| 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.