Binding affinity of lactose permease is not altered by the H+ electrochemical gradient
- Howard Hughes Medical Institute, Departments of Physiology and Microbiology, Immunology and Molecular Genetics, Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1662
-
Contributed by H. Ronald Kaback, July 9, 2004
Abstract
The x-ray structure of lactose permease of Escherichia coli (LacY) exhibits a single sugar-binding site at the apex of a hydrophilic cavity open to the cytoplasm, and it has been postulated
that the binding site has alternating access to either side of the membrane during turnover. Here, the affinity of LacY for
ligand in right-side-out or inside-out membrane vesicles is measured in the absence or presence of an H+ electrochemical gradient (
) by utilizing ligand protection against alkylation. Right-side-out or inside-out membrane vesicles containing LacY with a
single cysteine residue at position 148 exhibit K
D values for lactose or β-d-galactopyranosyl 1-thio-β-d-galactopyranoside of ≈1.0 mM or 40 μM, respectively, and no systematic change is observed in the presence of 
under conditions in which there is little or no accumulation of ligand. The results are consistent with a mechanism in which
the major effect of 
on sugar accumulation is caused by an increased rate of deprotonation on the inner face of the membrane, leading to an increase
in the rate of return of the unloaded symporter to the outer face of the membrane.
Footnotes
-
↵ * To whom correspondence should be addressed at: Howard Hughes Medical Institute/University of California, 5-748 Macdonald Research Laboratories, Box 951662, Los Angeles, CA 90095-1662. E-mail: ronaldk{at}hhmi.ucla.edu.
-
Abbreviations: LacY, lactose permease; MFS, major facilitator superfamily; TDG, β-d-galactopyranosyl 1-thio-β-d-galactopyransoside; NEM, N-ethylmaleimide; RSO, right-side out; ISO, inside out;
, H+ electrochemical gradient.
- Copyright © 2004, The National Academy of Sciences
