Unraveling the mechanism of the lactose permease of Escherichia coli

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.