Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli |
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Authors: | William R Trumble Paul V Viitanen Hemanta K Sarkar Mohindar S Poonian H Ronald Kaback |
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Institution: | 1. Roche Institute of Molecular Biology, Hoffmann-La Roche, Inc. Roche Research Center, Nutley, NJ 07110 USA;2. Department of Molecular Genetics Hoffmann-La Roche, Inc. Roche Research Center, Nutley, NJ 07110 USA |
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Abstract: | 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-β-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. |
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