Site-specific alteration of cysteine 176 and cysteine 234 in the lactose carrier of Escherichia coli.

In the present study, Cys-176 and Cys-234 in the lactose carrier have been modified to serine residues via site-specific mutagenesis. The resultant mutants have been characterized with regard to galactoside transport activity and sulfhydryl reagent sensitivity. The mutant proteins (in which Cys-176 or Cys-234 had been replaced with serine) are able to effectively transport galactosides, although the transport rates for lactose and methyl-beta-D-galactopyranoside are slightly reduced compared to the normal lactose carrier. In addition, both mutants are less sensitive than the wild-type to high concentrations of two different sulfhydryl reagents, N-ethylmaleimide and p-hydroxymercuribenzoate. Overall, the data are consistent with the idea that Cys-176 and Cys-234 are close to the substrate recognition site. However, neither residue appears to be essential for galactoside transport by providing an ionizable group near the active site or by forming a disulfide bond.