Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the {gamma}-L-glutamyl-L-cysteine-binding site |
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Authors: | Hara Takane; Tanaka Takuji; Kato Hiroaki; Nishioka Takaaki; Oda Jun'ichi |
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Affiliation: | Institute for Chemical Research, Kyoto University Uji, Kyoto 611, Japan |
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Abstract: | Lysl8, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathionesynthetase from Escherichia coli B are presumed to be highlyconcerned with the substrate, -L-glutamyl-L-cysteine ( -Glu-Cys),binding by X-ray crystallography and affinity labeling studies.Using site-directed mutagenesis, we investigated functionalroles of those residues for -Glu-Cys binding. The mutant enzymesof Arg86 and Asn283 altered their kinetic parameters, especiallythe Michaelis constants of -Glu-Cys. In the case of Asn283,the residue is not likely to have an essential role in -Glu-Cysbinding but its side chain would extend to make a van der Waalscontact with bound -Glu-Cys. Chemical modification of a cysteineresidue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86would not only be much responsible for -Glu-Cys binding butwould also have a role in maintaining the structural integrityof the enzyme. The other mutant enzymes showed little defectin their kinetic parameters of -Glu-Cys. |
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Keywords: | chemical modification/ glutathione synthetase/ site-directed mutagenesis/ substrate-binding site |
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