The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties.
The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H+, Na+ or Li(+)-sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar-carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.
Pourcher
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Laboratoire J. Maetz, Departement de Biologie du CEA, Villefranche sur Mer, France.
Bassilana
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Sarkar
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HK
Kaback
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Leblanc
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D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism*; D027981:Symporters
D004187:Disaccharides; D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D012964:Sodium