Properties of a mutant lactose carrier of <i>Escherichia coli</i> with a Cys¹⁴⁸ → Ser¹⁴⁸ substitution

The cysteine residue at position 148 in the lactose carrier protein of <i>Escherichia coli</i> has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the <i>lac</i>Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6–10 while the apparent affinity is reduced by a factor of 2–4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl<sub2</sub>, <i>p</i>-(chloro-mercuri)benzenesulfonate and <i>N</i>-ethylmaleimide) than in the wild type, and β-D-galactosyl 1-thio-β-D-galactoside does not protect the mutant carrier against slow inactivation by <i>N</i>-ethylmaleimide. It is concluded that the Cys¹⁴⁸ residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys¹⁴⁸→ Gly¹⁴⁸ replacement.