Probing the mechanism of a membrane transport protein with affinity inactivators.
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.
Guan
Lan
L
Howard Hughes Medical Institute, Department of Physiology, UCLA, Los Angeles, California 90095-1662, USA.
Sahin-Tóth
Miklós
M
Kálai
Tamás
T
Hideg
Kálmán
K
Kaback
H Ronald
HR
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
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