Chemical rescue of Asp237-->Ala and Lys358-->Ala mutants in the lactose permease of Escherichia coli.
Asp237 (helix VII) and Lys358 (helix XI) form a salt bridge in the lactose permease, and neutral replacement of either residue inactivates. Remarkably, noncovalent neutralization of the unpaired Asp or Lys residue, respectively, with n-alkylsulfonates or n-alkylamines of appropriate size restores active transport to high levels in the mutants. Saturation with respect to the concentration of the alkylamines and different size preferences suggest that the alkylamines bind sterically at position 358. Rescue of Asp237-->Ala by alkylsulfonates is apparently more indiscriminate, since methane-, ethane-, or propane-sulfonate have comparable effects. Sodium and chloride, respectively, are also effective in rescuing the Lys358-->Ala and Asp237-->Ala mutants, while various other compounds are ineffective. In marked contrast to Asp237-->Ala or Lys358-->Ala permease, alkylsulfonates or alkylamines have no effect whatsoever on the activity of mutants with neutral replacements for Asp240, Glu269, Arg302, Lys319, His322, or Glu325. The results support the conclusion that neutral replacement of one member of the charge pair between Asp237 and Lys358 leads to inactivation because of an unpaired 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 either Asp237 or Lys358 or the salt bridge between the two residues.
Frillingos
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Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1570, USA.
Kaback
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D000588:Amines / Q000494:pharmacology; D001665:Binding Sites; D001693:Biological Transport, Active; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007477:Ions; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins; D017354:Point Mutation; D017433:Protein Structure, Secondary; D012965:Sodium Chloride / Q000494:pharmacology; D013451:Sulfonic Acids / Q000494:pharmacology; D027981:Symporters
D000588:Amines; D029968:Escherichia coli Proteins; D007477:Ions; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013451:Sulfonic Acids; D027981:Symporters; D012965:Sodium Chloride; C021499:lactose permease; D007785:Lactose