Cystamine-Sepharose. A probe for the active site of gamma-glutamylcysteine synthetase.
gamma-Glutamylcysteine synthetase, previously known to be potently inhibited by cystamine, has been found to bind covalently to cystamine-Sepharose. ATP facilitates, whereas glutamate plus magnesium ions inhibit, binding of the enzyme to cystamine-Sepharose. A large fraction of the enzyme applied to columns of cystamine-Sepharose binds by forming a disulfide bond between cysteamine-Sepharose and a sulfhydryl group at or near the active site of the enzyme. The enzyme may be released by treatment with dithiothreitol. Some of the enzyme applied to such columns is inactivated and not bound covalently to the column. That the enzyme does not bind to columns of S-(S-methyl)cysteamine-Sepharose, whereas free S-(S-methyl)cysteamine is a potent inhibitor, indicates that a cysteamine-S disulfide moiety derived from the external cysteamine residue of cystamine-Sepharose is the critical group recognized by the enzyme. The observed partitioning of the enzyme on columns of cystamine-Sepharose between covalently column-bound enzyme and nonbound inactivated enzyme suggests that the reactive enzyme sulfhydryl group forms a disulfide linkage with the sulfur atom at the immobilized end of cystamine to link the enzyme to the column and to liberate free cysteamine, and also that the enzyme interacts with the external cysteamine moiety of the bound cystamine. The latter may occur if the free cysteamine released is spontaneously oxidized to free cystamine followed by its inhibition of the enzyme, or if there is a direct reaction between the enzyme-reactive sulfhydryl group and the sulfur atom of the external cysteamine moiety of cystamine-Sepharose.
D000818:Animals; D001665:Binding Sites; D003538:Cystamine / Q000494:pharmacology*; D005721:Glutamate-Cysteine Ligase / Q000302:isolation & purification / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010453:Peptide Synthases / Q000378:metabolism*; D011134:Polysaccharides; D011485:Protein Binding; D051381:Rats; D012685:Sepharose / Q000031:analogs & derivatives
D011134:Polysaccharides; C034391:cystamine-sepharose; D012685:Sepharose; D010453:Peptide Synthases; D005721:Glutamate-Cysteine Ligase; D003538:Cystamine