SULFHYDRYL OXIDATION OF MUTANTS WITH CYSTEINE IN-PLACE OF ACIDIC RESIDUES IN THE LACTOSE PERMEASE

To examine further the role of charge-pair interactions in the structu re and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIlI), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues. Each mutant was then oxidized with H2O2 in ord er to generate a sulfinic and/or sulfonic acid at these positions. Due to the isosteric relationship between aspartate and sulfinate, in par ticular, and the lower pK(a) of the sulfinic and sulfonic acid side ch ains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates. Asp237-->Cys or Asp240-->Cys permease is inactiv e, as shown previously, but H2O2 oxidation restores activity to an ext ent similar to that observed when a negative charge is reintroduced by other means. Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport. The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu12 6, Glu269, and Glu325 are irreplaceable. Although Glu269-->Cys permeas e does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranosi de, a property observed with Glu269-->Asp permease. The observation su pports the idea that an acidic residue at position 269 is important fo r substrate recognition. Finally, oxidized Glu325-->Cys permease catal yzes equilibrium exchange with an apparent pK(a) of about 6.5, more th an a pH unit lower than that observed with Glu325-->Asp permease, ther eby providing strong confirmatory evidence that a negative charge at p osition 325 determines the rate of translocation of the ternary comple x between the permease, substrate, and H+.