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 Ala122→Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (Δμ̄H+) markedly increases the rate of reaction between Cys122 and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala122→Cys mutation is combined with a mutation (Cys154→Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and Δμ̄H+ has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala122 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.
CITATION STYLE
Guan, L., Sahin-Tóth, M., Kálai, T., Hideg, K., & Kaback, H. R. (2003). Probing the mechanism of a membrane transport protein with affinity inactivators. Journal of Biological Chemistry, 278(12), 10641–10648. https://doi.org/10.1074/jbc.M211355200
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