Probing the mechanism of a membrane transport protein with affinity inactivators

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 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.