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.