@article{ref1,
title="Probing the mechanism of a membrane transport protein with affinity inactivators",
journal="Journal of biological chemistry",
year="2003",
author="Guan, Lan and Sahin-Tóth, Miklós and Kálai, Tamás and Hideg, Kálmán and Kaback, H. Ronald",
volume="278",
number="12",
pages="10641-10648",
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.<p /><p>Language: en</p>",
language="en",
issn="0021-9258",
doi="10.1074/jbc.M211355200",
url="http://dx.doi.org/10.1074/jbc.M211355200"
}