Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis.
Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents.
D000409:Alanine; D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003062:Codon; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D005798:Genes, Bacterial; D005981:Glutathione Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010453:Peptide Synthases / Q000378:metabolism*; D010957:Plasmids; D013438:Sulfhydryl Compounds; D013861:Thiocyanates / Q000494:pharmacology; D014169:Transformation, Bacterial; D020245:p-Chloromercuribenzoic Acid
D000596:Amino Acids; D002729:Chloromercuribenzoates; D003062:Codon; D013438:Sulfhydryl Compounds; D013861:Thiocyanates; D020245:p-Chloromercuribenzoic Acid; D004228:Dithionitrobenzoic Acid; D010453:Peptide Synthases; D005981:Glutathione Synthase; D003545:Cysteine; D005033:Ethylmaleimide; C008658:2-nitro-5-thiocyanobenzoic acid; D000409:Alanine