title: Unraveling the mechanism of the lactose permease of Escherichia coli.
abstract: We studied the effect of pH on ligand binding in wild-type lactose permease or mutants in the four residues-Glu-269, Arg-302, His-322, and Glu-325-that are the key participants in H(+) translocation and coupling between sugar and H(+) translocation. Although wild-type permease or mutants in Glu-325 and Arg-302 exhibit marked decreases in affinity at alkaline pH, mutants in either His-322 or Glu-269 do not titrate. The results offer a mechanistic model for lactose/H(+) symport. In the ground state, the permease is protonated, the H(+) is shared between His-322 and Glu-269, Glu-325 is charge-paired with Arg-302, and substrate is bound with high affinity at the outside surface. Substrate binding induces a conformational change that leads to transfer of the H(+) from His-322/Glu-269 to Glu-325 and reorientation of the binding site to the inner surface with a decrease in affinity. Glu-325 then is deprotonated on the inside because of rejuxtaposition with Arg-302. The His-322/Glu-269 complex then is reprotonated from the outside surface to reinitiate the cycle.
authors.affiliation: Howard Hughes Medical Institute, Department of Physiology and Microbiology, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662, USA.
authors.forename: M
authors.initials: M
authors.lastname: Sahin-Tóth
authors.forename: A
authors.initials: A
authors.lastname: Karlin
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D017354:Point Mutation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D027981:Symporters
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease