CHEMICAL RESCUE OF ASP237-]ALA AND LYS358-]ALA MUTANTS IN THE LACTOSEPERMEASE OF ESCHERICHIA-COLI

Asp237 (helix VII) and Lys358 (helix XI) form a salt bridge in the lac tose permease, and neutral replacement of either residue inactivates. Remarkably, noncovalent neutralization of the unpaired Asp or Lys resi due, respectively, with n-alkylsulfonates or n-alkylamines of appropri ate size restores active transport to high levels in the mutants. Satu ration with respect to the concentration of the alkylamines and differ ent size preferences suggest that the alkylamines bind sterically at p osition 358. Rescue of Asp237-->Ala by alkylsulfonates is apparently m ore indiscriminate, since methane-, ethane-, or propane-sulfonate have comparable effects. Sodium and chloride, respectively, are also effec tive in rescuing the Lys358-->Ala and Asp237-->Ala mutants, while vari ous other compounds are ineffective. In marked contrast to Asp237-->Al a or Lys358-->Ala permease, alkylsulfonates or alkylamines have no eff ect whatsoever on the activity of mutants with neutral replacements fo r Asp240, Glu269, Arg302, Lys319, His322, or Glu325. The results suppo rt the conclusion that neutral replacement of one member of the charge pair between Asp237 and Lys358 leads to inactivation because of an un paired charge in the low dielectric of the membrane. In addition, the findings are consistent with the idea that interactions between Arg302 and Glu325, His 322 and Glu269, and Asp240 and Lys319 play important roles in the mechanism of the permease, which is not the case for eith er Asp237 or Lys358 or the salt bridge between the two residues.