title: 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 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Physiology, UCLA, Los Angeles, California 90095-1662, USA.
authors.forename: Lan
authors.initials: L
authors.lastname: Guan
authors.forename: Miklós
authors.initials: M
authors.lastname: Sahin-Tóth
authors.forename: Tamás
authors.initials: T
authors.lastname: Kálai
authors.forename: Kálmán
authors.initials: K
authors.lastname: Hideg
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D000345:Affinity Labels; D002384:Catalysis; D003545:Cysteine; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008698:Mesylates / Q000737:chemistry / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D021381:Protein Transport; D027981:Symporters
chemical_list: D000345:Affinity Labels; D029968:Escherichia coli Proteins; D005697:Galactosides; D005960:Glucosides; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008698:Mesylates; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C074502:methanethiosulfonate; C021499:lactose permease; D003545:Cysteine