Identification of an essential cysteine residue in human glutathione synthase.
Glutathione is essential for a variety of cellular functions, and is synthesized from gamma-glutamylcysteine and glycine by the action of glutathione synthase (EC 6.3.2.3). Human glutathione synthase is a dimer of two identical subunits, each composed of 474 amino acids. Little is known about the structure-function relationships of mammalian glutathione synthases and, in order to gain a greater understanding of this critical enzyme, we have probed the role of cysteine residues by chemical modification and site-directed mutagenesis. Preincubation with thiol reagents such as p-chloromercuribenzoate, N-ethylmaleimide, iodoacetate and 5,5'-dithiobis-(2-nitrobenzoate) resulted in significant inhibition of recombinant human glutathione synthase. Each subunit contains cysteine residues at positions 294, 409 and 422, and we have prepared four different mutants by replacing individual cysteine residues, or all of the cysteine residues, with alanine. The C294A and C409A cysteine mutants retained significant residual activity, indicating that these two cysteine residues are not essential for activity. In contrast, substantial decreases in enzymic activity were detected with the C422A and cysteine-free mutants. This suggests that Cys-422 may play a significant structural or functional role in human glutathione synthase.
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Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra, Australia.
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D003545:Cysteine / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D005981:Glutathione Synthase / Q000737:chemistry*; D006801:Humans; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D016297:Mutagenesis, Site-Directed; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000494:pharmacology
D007461:Iodoacetates; C036590:1-(1-phenylcyclohexyl)-3-methylpiperidine; D004228:Dithionitrobenzoic Acid; D005981:Glutathione Synthase; D010622:Phencyclidine; D003545:Cysteine; D005033:Ethylmaleimide; D019807:Iodoacetic Acid