Abstract
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.
Highlights
We demtates, whereas glutamate plus magnesium ions inhibit, onstrate here thaty-glutamylcysteine synthetase binds covabinding of the enzyme to cystamine-Sepharose.A large lently to a modified cystamine which is immobilized through fraction of the enzyme appliedto columns of cystamine- one amino group by attachment to a large Sepharose bead, Sepharose binds by forming a disulfide bond between and that such binding is significantly facilitated by the prescysteamine-Sepharose and a sulfhydryl group at or near theactive site of the enzyme
The enzyme does not bind to inhibitor, indicates that a cysteamine-S disulfide moi- S-(S-methyl)cysteamine, immobilized by attachety derived from the external cysteamine residue of ment through its only amino group to Sepharose, a finding cystamine-Sepharose is the critical group recognized which provides insight into the nature of the interaction of by the enzyme
On columns of cystamine-Sepharose between covalently column-bound enzymaend nonboundinactivated enzyme suggests that the reactive enzyme sulfhydryl Materials and Methods-Activated CH-Sepharose 4B
Summary
From the Departmentof Biochemistry, Cornel University Medical College, New York, New York 10021. Be potently inhibited by cystamine, has been found to suggested that cystamine may uniquely fulfirather stringent bind covalently to cystamine-Sepharose. LGlutamate andmagnesium ions, which enhance the recovery of the enzymatic activity retrieved from the cystamine-Sepharose column, protect the enzyme against inhibition by both rose, whereas free S-(S-methy1)cysteamineis a potent free and immobilizedcystamine. The enzyme does not bind to inhibitor, indicates that a cysteamine-S disulfide moi- S-(S-methyl)cysteamine, immobilized by attachety derived from the external cysteamine residue of ment through its only amino group to Sepharose, a finding cystamine-Sepharose is the critical group recognized which provides insight into the nature of the interaction of by the enzyme. On columns of cystamine-Sepharose between covalently column-bound enzymaend nonboundinactivated
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