Ser57 in the Na+/proline permease of Escherichia coli is critical for high-affinity proline uptake

Autor(en): Quick, M
Tebbe, S
Jung, H
Stichwörter: Biochemistry & Molecular Biology; COTRANSPORT; GENE; IDENTIFICATION; PROTEIN; PUTP; RECONSTITUTION; secondary transport; site-directed mutagenesis; SITE-SPECIFIC ALTERATION; SUBSTRATE-SPECIFICITY; SYMPORT CARRIER; TRANSPORT
Erscheinungsdatum: 1996
Herausgeber: SPRINGER VERLAG
Journal: EUROPEAN JOURNAL OF BIOCHEMISTRY
Volumen: 239
Ausgabe: 3
Startseite: 732
Seitenende: 736
Zusammenfassung: 
Ser57 in the Na+/proline permease of Escherichia coli has been replaced with alanine, cysteine. glycine, or threonine, and properties of the corresponding putP mutants have been analyzed. Although Ser57 Is not essential for activity, the amino acid side chain at this position is critical for proline uptake. Thus alanine, cystein, glycine, or threonine in place of Ser57 reduces the initial rate of proline transport under standard conditions to less than 10% of the wild-type value. In addition. substitution of Ser57 in the Na+/proline permease reduces the sensitivity of E. coli cells to the toxic proline analogs L-azrtidine-2-carboxylate and 3,4-dehydro-D,L-proline, Replacement of Ser57 with alanine or cysteine results in apparent affinities for proline that are reduced by more than two orders of magnitude, and permeases with threonine and glycine in place of Ser57 yield apparent affinities reduced by a factor of 60 and 18, respectively, relative to wild-type. In contrast, all of the Ser57 replacements analyzed cause only small changes in V-max values. All permease molecules containing Ser57 substitutions are inserted into the membrane in amounts comparable to the wild-type protein as shown by immunoblot analysis. These results indicate that alterations of proline transport and sensitivity to toxic proline analogs have to be attributed primarily to defects in substrate binding. It is suggested that the serine residue at position 57 of the permease is located within the substrate-binding domain of the protein.
ISSN: 00142956
DOI: 10.1111/j.1432-1033.1996.0732u.x

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