Citation

Guan L, Sahin-Tóth M, Kálai T, Hideg K, Kaback HR. J. Biol. Chem. 2003; 278(12): 10641-10648.

Copyright

(Copyright © 2003, American Society for Biochemistry and Molecular Biology)

DOI

10.1074/jbc.M211355200

PMID

12471022

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.

Language: en

Keywords

Affinity Labels; Catalysis; Cysteine; Escherichia coli; Escherichia coli Proteins; Galactosides; Glucosides; Membrane Transport Proteins; Mesylates; Monosaccharide Transport Proteins; Protein Transport; Symporters