Melibiose permease of Escherichia coli: mutation of aspartic acid 55 in putative helix II abolishes activation of sugar binding by Na+ ions.
An aspartic residue (Asp55) located in the putative transmembrane alpha-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient. (3H) p-nitrophenyl-alpha-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na(+)-binding site and 2) that the cation and sugar binding sites may be overlapping.
Pourcher
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Laboratoire J. Maetz, Département de Biologie Cellulaire et Moléculaire du Commissariat à l'Energie Atomique, Villefranche sur mer, France.
Deckert
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Bassilana
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Leblanc
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D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009598:Nitrophenylgalactosides / Q000378:metabolism; D015345:Oligonucleotide Probes; D010957:Plasmids; D012964:Sodium / Q000494:pharmacology*; D027981:Symporters
D026901:Membrane Transport Proteins; D015345:Oligonucleotide Probes; D027981:Symporters; D009598:Nitrophenylgalactosides; D001224:Aspartic Acid; C059200:4-nitrophenylgalactoside; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C021548:melibiose permease; D008553:Melibiose; D012964:Sodium