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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.
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