Site-directed mutagenesis of cysteine-148 in the lac permease of Escherichia coli: effect on transport, binding, and sulfhydryl inactivation.
By subjecting the lac y gene of Escherichia coli to oligonucleotide-directed, site-specific mutagenesis, Cys148 in the lac permease has been replaced with a Gly residue [Trumble, W. R., Viitanen, P. V., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860]. Recombinant plasmids bearing wild-type or mutated lac y were constructed and used to transform E. coli T184. Steady-state levels of lactose accumulation, the apparent Km for lactose under energized conditions, and the KD for p-nitrophenyl alpha-D-galactopyranoside are comparable in right-side-out vesicles containing wild-type or mutant permease. In contrast, the Vmax for lactose transport in vesicles containing mutant permease is significantly decreased. Although antibody binding studies reveal that vesicles from the mutant contain almost as much permease as wild-type vesicles, surprisingly only about one-fourth of the altered molecules bind p-nitrophenyl alpha-D-galactopyranoside with high affinity. Mutant permease is less sensitive to inactivation by N-ethylmaleimide, although the alkylating agent is still capable of completely inhibiting transport activity. Importantly, beta-galactosyl 1-thio-beta-D-galactopyranoside affords complete protection of wild-type permease against N-ethylmaleimide but has no protective effect whatsoever in the mutant. The rate of inactivation of wild-type and mutant permeases by N-ethylmaleimide is increased at alkaline pH and by the presence of a proton electrochemical gradient (interior negative and alkaline), and these phenomena are exaggerated in vesicles containing mutant permease. Finally, p-(chloromercuri)benzenesulfonate, which completely displaces bound p-nitrophenyl alpha-D-galactopyranoside from wild-type permease, does not affect binding in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide