PMID:8577699
| Citation |
Hara, T, Tanaka, T, Kato, H, Nishioka, T and Oda, J (1995) Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the gamma-L-glutamyl-L-cysteine-binding site. Protein Eng. 8:711-6 |
|---|---|
| Abstract |
Lys18, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathione synthetase from Escherichia coli B are presumed to be highly concerned with the substrate, gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), binding by X-ray crystallography and affinity labeling studies. Using site-directed mutagenesis, we investigated functional roles of those residues for gamma-Glu-Cys binding. The mutant enzymes of Arg86 and Asn283 altered their kinetic parameters, especially the Michaelis constants of gamma-Glu-Cys. In the case of Asn283, the residue is not likely to have an essential role in gamma-Glu-Cys binding but its side chain would extend to make a van der Waals contact with bound gamma-Glu-Cys. Chemical modification of a cysteine residue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86 would not only be much responsible for gamma-Glu-Cys binding but would also have a role in maintaining the structural integrity of the enzyme. The other mutant enzymes showed little defect in their kinetic parameters of gamma-Glu-Cys. |
| Links | |
| Keywords |
Base Sequence; Binding Sites; Cysteine/chemistry; DNA Primers; Dipeptides/metabolism; Dithionitrobenzoic Acid/chemistry; Escherichia coli/enzymology; Glutathione Synthase/genetics; Glutathione Synthase/metabolism; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed |
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