Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the gamma-L-glutamyl-L-cysteine-binding site.
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.
Hara
T
T
Institute for Chemical Research, Kyoto University, Japan.
Tanaka
T
T
Kato
H
H
Nishioka
T
T
Oda
J
J
D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers; D004151:Dipeptides / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005981:Glutathione Synthase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D017931:DNA Primers; D004151:Dipeptides; D004228:Dithionitrobenzoic Acid; D005981:Glutathione Synthase; D003545:Cysteine; C017341:gamma-glutamylcysteine