Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli.
The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-beta-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose: proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.
D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D005796:Genes / Q000187:drug effects; D005798:Genes, Bacterial / Q000187:drug effects; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics*; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D027981:Symporters; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D027981:Symporters; C021499:lactose permease; D007785:Lactose; D005033:Ethylmaleimide