Arg-302 facilitates deprotonation of Glu-325 in the transport mechanism ofthe lactose permease from Escherichia coli

A mechanistic model for lactose/H+ symport via the lactose permease of Esch erichia coli proposed recently indicates that the permease must be protonat ed to bind ligand with high affinity. Moreover, in the ground state, the sy mported H+ is shared between His-322 (helix X) and Glu-269 (helix VIII), wh ereas Glu-325 (helix X) is charge-paired with Arg-302 (helix IX). Substrate binding at the outer surface induces a conformational change that leads to transfer of the H+ to Glu-325 and reorientation of the binding site to the inner surface. After release of the substrate, Glu-325 is deprotonated on the inside because of rejuxtapositioning with Arg-302. To test the role of Arg-302 in the mechanism, the catalytic properties of mutants Arg-302 --> A la and Arg-302 --> Ser were studied. Both mutants are severely defective in active lactose transport, as well as in efflux or influx down a concentrat ion gradient, translocation modes that involve net H+ movement. In marked c ontrast, the mutants catalyze equilibrium exchange of lactose and bind liga nd with high affinity. These characteristics are remarkably analogous to th ose of permease mutants with neutral replacements for Glu-325, a residue th at plays a direct role in H+ translocation. Th se observations lend strong support for the argument that Arg-302 interacts with Glu-325 to facilitate deprotonation of the carboxylic acid during turnover.