Gamma-glutamylcysteine synthetase. Interactions of an essential sulfhydryl group.
gamma-Glutamylcysteine synthetase (isolated from rat kidney) has one sulfhydryl group that reacts with 5,5'-dithiobis-(2-nitrobenzoate). This single exposed sulfhydryl group is not required for enzyme activity. The enzyme is potently inactivated by cystamine, which apparently interacts with a sulfhydryl group at the active site to form a mixed disulfide. 5,5'-Dithiobis-(2-nitrobenzoate) does not interact with the sulfhydryl group that reacts with cystamine. After the enzyme was 90% inactivated by reaction with cystamine, 3.4 mol of 5,5'-dithiobis-(2-nitrobenzoate) reacted per mol of enzyme, indicating that binding of cystamine exposes sulfhydryl groups which are apparently buried or unreactive in the native enzyme. L-Glutamate (but not D-glutamate or L-alpha-aminobutyrate) protected against inactivation by cystamine. In contrast, ATP enhanced the rate of inactivation by cystamine, and the apparent Km value for this effect is similar to that for ATP in the catalytic reaction. Studies on the structural features of cystamine that facilitate its interaction with the enzyme showed that selenocystamine, monodansylcystamine, and N-[2[2-aminoethyl)-dithio)ethyl]-4-azido-2-nitrobenzeneamine are also good inhibitors. Whereas S-(S-methyl)cysteamine-Sepharose does not interact with the enzyme (Seelig, G. F., and Meister, A. (1982) J. Biol. Chem. 257, 5092-5096), S-(S-methyl)cysteamine is a potent inhibitor; 1 mol of this compound completely inactivated 1 mol of enzyme. In the course of this work, a useful modification of the method for isolating this enzyme from kidney was developed.
D000818:Animals; D001665:Binding Sites; D003538:Cystamine / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D010453:Peptide Synthases; D005721:Glutamate-Cysteine Ligase; D003538:Cystamine