Cystamine-Sepharose A PROBE FOR THE ACTIVE SITE OF y-GLUTAMYLCYSTEINE SYNTHETASE*

y-Glutamylcysteine synthetase, previously known to be potently inhibited by cystamine, has been found to bind covalently to cystamine-Sepharose. ATP facili- tates, 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-methy1)cysteamine-Sepha- rose, whereas free S-(S-methy1)cysteamine is a potent inhibitor, indicates that a cysteamine-S disulfide moi- ety 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 cova- lently 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,

y-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-methy1)cysteamine-Sepharose, whereas free S-(S-methy1)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.
y-Glutamylcysteine synthetase, which catalyzes the first step in the synthesis of glutathione (Reaction 1 (1, 2)) is very potently inhibited by the disulfide cystamine (3-5). Treatment of the inhibited enzyme with reducing agents reverses the in-L-glutamate + L-cysteine + ATP e Mg2+ (1) L-y-glutamyl-L-cysteine + ADP + Pi hibition. This observation and other data indicate that interaction between the enzyme and cystamine leads to the formation of a mixed disulfide between cysteamine and an enzyme sulfhydryl group (3, 5). The inability of certain cysta-* This work was suppoxed in part by a grant from the United States Public Health Service, National Institutes of Health. A preliminary report of some of these findings was presented at the 72nd Annual Meeting of the American Society of Biological Chemists ((1981) Fed. Proc. 40, 1833 (abstract 1692)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mine analogs and other disulfides to inhibit the enzyme (3) suggested that cystamine may uniquely fulfi rather stringent requirements for inhibition of this enzyme. However, we demonstrate here that y-glutamylcysteine synthetase binds covalently to a modified cystamine which is immobilized through one amino group by attachment to a large Sepharose bead, and that such binding is significantly facilitated by the presence of ATP. The immobilized enzyme can subsequently be cleaved from the matrix by treatment with dithiothreitol. L-Glutamate and magnesium ions, which enhance the recovery of the enzymatic activity retrieved from the cystamine-Sepharose column, protect the enzyme against inhibition by both free and immobilized cystamine. The enzyme does not bind to S-(S-methyl)cysteamine, similarly immobilized by attachment through its only amino group to Sepharose, a finding which provides insight into the nature of the interaction of the enzyme with cystamine.

EXPERIMENTAL PROCEDURES
Materials and Methods-Activated CH-Sepharose 4B (6-carbon spacer arm) was obtained from Pharmacia. Sodium dodecyl sulfate was obtained from BDH Biochemicals, and acrylamide was purchased from Bio-Rad. The substrates, buffers, cystamine, 5,5'-dithio(bis)nitrobenzoic acid, dithiothreitol, and cysteamine-agarose, were obtained from Sigma. y-Glutamylcysteine synthetase was purified (6) from frozen rat kidneys (Pel-Freeze) and kidneys of rats purchased from Taconic Farms; the purified preparations gave a single band on gel electrophoresis (6) and had specific activities in the range of 900-1400 units/mg.
Preparation of Cystamine-Sepharose-CH-Sepharose 4B (5 g) was suspended in 1 m~ HCl to give 15 ml of swelled gel. The gel was rinsed with 1 liter of 1 mM HCI. A 10-fold excess of ligand (3.5 g of cystamine) was dissolved in 25 ml of 0.1 M NaHC03 buffer containing 0.5 M NaCl (coupling buffer) and mixed in an Erlenmeyer flask with the washed gel. The flask was shaken gently at 4 "C overnight. Excess ligand was washed from the gel with 1 liter of coupling buffer, and any remaining active groups were blocked by washing with 1 M ethanolamine at pH 9.0. The resin WPS then washed successively with 300 ml of 0.1 M sodium acetate buffer (pH 4.0) containing I M NaC1, and 300 ml of 0.1 M Tris-HC1 buffer (pH 8.0) containing 1 M NaCl for a total of three times each, and then stored in the Tris buffer. The amount of bound cystamine (quantitated after reduction by reaction with 5,5'-dithio(bis)nitrobenzoic acid) was estimated to be about 1.8 pmol/ml. Prior to use, the cystamine-Sepharose was equilibrated with 10 mM imidazole buffer (pH 8.2).
Preparation of S-(S-Methyl)cysteamine-Sepharose-Cysteamineagarose (2.0 m l ) was washed extensively with water. It was then suspended in a mixture containing 400 pl of water, 400 p1 of methanol, and 40 pl of methyl methanethiosulfonate, and allowed to react at 24 "C for 30-45 min. It was washed again with copious amounts of water to remove excess reagents, and was then equilibrated in 10 mM imidazole buffer (pH 8.2). The amount of bound S-(S-methy1)cysteamine, determined by reaction with 5,5"dithio(bis)nitrobenzoic acid before and after reductive cleavage of the disulfide, was estimated to be 0. Gel Electrophoresis-Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was performed by the method of Weber and Osborn (9) using 7.5% gels. Gels were polymerized in 6 M urea (10) and run at 5 mA/tube or a total of 55 mA/slab gel. Samples were mixed with an equal volume of saturated sucrose containing bromphenol blue tracking dye. Protein bands were detected by staining with Coomassie blue (9). Quantitation of protein bands was performed on a Cary/Varian 210 spectrophotometer equipped with a gel scanning accessory.

Effect of Substrates on Inactiuation of y-Glutamylcysteine
Synthetase by Cystamine-Incubation of the enzyme with 162 p~ cystamine at 4 "C for 30 min led to a loss of about 60% of the initial activity ( Table I). Addition of ATP to the preincubation mixture led to complete loss of enzyme activity under these conditions.' On the other hand, the addition of magnesium chloride and L-glutamate (separately or together) partially protected against inactivation, probably because cystamine binds at or close to the glutamate binding site of the enzyme where it reacts with an enzyme sulfhydryl group (5). L-a-Aminobutyrate did not protect the enzyme against inactivation by cystamine. When the enzyme samples inhibited by preincubation with cystamine under the conditions given in Table I were subsequently treated with 50 m~ dithiothreitol, there was virtually complete restoration of enzyme activity.
Chromatography of Purified y-Glutamylcysteine Synthetase on Cystamine-Sepharose-In these studies we found that the enzyme is effectively bound by cystamine-Sepharose and that preincubation of the enzyme with ATP markedly increases binding. Fig. lA illustrates a typical chromatography profie. In this study, the enzyme was preincubated with ATP and then applied to a column of cystamine-Sepharose. Some protein eluted with the starting buffer and a larger amount eluted with 0.5 M sodium chloride ( Fig. l A , arrow a). Enzyme activity equivalent to about 60% of that applied remained covalently bound; this was released by elution with a buffer containing 50 mM dithiothreitol, 10 m~ magnesium chloride, and 10 m~ L-glutamate (arrow b). The noncovalently bound protein, which eluted with 0.5 M sodium chloride was inactive but could be completely reactivated by treatment with dithiothreitol. The total recovery of the applied enzyme activity in this chromatography was about 90%. Similar results were obtained when this experiment (Fig. lA) was done with a preincubation mixture that contained 5 mM manganese chloride in addition to ATP. Fig. 1 B shows that the covalent interaction between enzyme and cystamine-Sepharose is substantially diminished when the enzyme is not preincubated with ATP. Most of the protein does not bind covalently to the column under these conditions; about 87% of the activity was eluted with a buffer containing 0.5 M sodium chloride (Fig. lB, arrow a). After treatment of this fraction with dithiothreitol, an equivalent amount of enzyme activity was found. About 16% of the applied enzyme activity became covalently linked to the column in this study; this was eluted with buffer containing dithiothreitol, magne-' Although this effect of ATP was observed in the absence of added magnesium ions, it is likely that magnesium ions used in the purification are present in the enzyme preparation. The kinetics of the effect of ATP on cystamine inactivation will be addressed in detail in a subsequent paper. a Pseudo-first order rate constant (X min" for inactivation of enzyme. y-Glutamylcysteine synthetase (12.8 units) was incubated at 4 "C in 10 mM imidazole buffer (pH 8.2) containing 162 PM cystamine and the compounds indicated above (total volume, 120 pl). After 30 min, 10-pl portion.: were removed (containing 1.06 unit of enzyme and 13.5 pmol of cystamine), and assayed at 37 "C in reaction mixtures con- ' Controls were treated in an identical manner except cystamine was omitted. Each of the controls retained 100% of the initial activity. sium chloride, and L-glutamate (Fig. lB, arrow b). The study described in Fig. 1C illustrates that magnesium ions and Lglutamate facilitate the recovery of enzyme activity. In this study, about 38% of the enzyme was eluted with buffer containing 0.5 M sodium chloride (Fig. IC, arrow a). The buffer was then changed to include dithiothreitol (Fig. IC, arrow e); this led to release of about 11% of the activity. On subsequent application of a buffer containing magnesium chloride and Lglutamate as well as dithiothreitol (Fig. IC, arrow b), substantial additional enzyme activity was released.
When the enzyme was preincubated with mangesium chloride and L-glutamate and when these comFocnds were included in the starting buffer, the enzyme did not bind covalently to the column. Thus, as shown in Fig. lD, virtually all of the enzyme activity applied appeared early in the effluent. These findings are consistent with the observation that glutamate plus magnesium ions effectively protects against inactivation of the enzyme by cystamine (Table I). When dithiothreitol was added to the starting buffer there was also no covalent interaction between the enzyme and cystamine-Sepharose; the enzyme eluted early in the profiie in a manner identical with that shown in Fig. 1D. In the study described in Fig. lE, addition of 10 m~ cystamine to the buffer (arrow d) was followed by elution of about 30% of the enzyme. This effect, which does not represent a displacement of covalently bound enzyme, appears to constitute nonspecific elution of noncovalently bound enzyme. At arrow e, the buffer was changed to include 50 m~ cystamine; this did not elute further protein. However, when a buffer containing dithiothreitol, magnesium chloride, and L-glutamate was applied (Fig. lE, arrow b), elution of covalently bound enzyme (as in Figs. lA and C) occurred.
The findings given above indicate that a large fraction of the enzyme applied to cystamine-Sepharose binds covalently by forming a mixed disulfide between an enzyme sulfhydryl group and a bound cysteamine residue. Some of the applied enzyme is noncovalently bound and is released in an inactive form; this can be reactivated by treatment with dithiothreitol indicating that this portion of the enzyme has formed a mixed disulfide with the external cysteamine residue of the cystamine-Sepharose.
Chromatography of Purified y-Ghtamylcysteine Synthe- with either a buffer containing NaCl (Fig. 2, arrow a ) or a cystamine-Sepharose. A, a 500-pl portion (67.5 units) of the enzyme was incubated for 10 min with 1 m~ ATP. The mixture was then Chromatography of Impure y-Glutamylcysteine Synthetase applied to a column (0.75 x 4.1 cm) which had previously been on Cystamine-Sepharose-The potentid Of cystaequilibrated with 10 mM imidazole (pH 8.2) buffer with a flow rate of mine-Sepharose for purification of the enzyme was explored 40 ml/hr. At arrow a, the buffer was changed to include 0.5 M NaCI. using enzyme purified through step 4 of the isolation proce-At arrow b, the buffer was changed to 10 mM imidazole buffer dure (6); this material contains about 10% active enzyme. A containing 50 m~ dithiothreitol, 10 mM L-glutamate, and 10 m~ solution of the enzyme in 10imidazole buffer ( P~ 8.2) MgC12. B, a 500-pl portion (352 units) of the enzyme was applied to a column (0.75 X 4.2 cm) which had been equilibrated with 10 mM was applied to a column containing 2 ml of cystamine-Sephimidazole buffer ( p~ 8.2) with a flow rate of 40 d/b. The buffer arose previously equilibrated with the Same buffer. Fig. 3 changes were identical with those in A. C, a 500-pl portion (67 units) shows the chromatography profile obtained; about 10% of of enzyme was incubated for 10 min with 0.98 mM ATP and applied nonactive protein did not bind, but eluted with the starting to a column (0.75 x 4.1 cm) previously equilibrated with 10 mM buffer. Buffer containing 0.5 M NaCl (Fig. 3, arrow a) eluted imidazole buffer (pH 8.2) with a flow rate Of 40 &hr. At arrow a, additional protein and activity. Elution with dithiothreitol, the buffer was changed to include 0.5 M NaCl. At arrow c, the buffer magnesium chloride, and L-glutamate (Fig. 3,  b) led to was changed to 10 m~ imidazole buffer containing 50 m~ dithiothreitol. At b, 10 imidazole buffer containing 50 mM dithio-prompt appearance of about 50% of the initial activity applied threitol, 10 mM L-glutamate, and 10 mM MgClz was used. 0, this is a to the Column. The total recovery of enzyme activity was 90%; typical elution profile for enzyme that had been preincubated with the material eluted from the column (Fig. 3, arrow b) was either (a) 47.6 m~ dithiothreitol and then applied to a column (0.75 about 70% pure as determined by spectrophotometric analysis X 3.0 cm) previously equilibrated with 10 mM imidazole (pH 8.2) with a flow rate of 60 d / h , or ( 6 ) L-glutamate (8.3 mM) and 8.3 mM MgCL and then applied to a column (0.75 X 3.2 cm) previously equilibrated 2 S-(S-Methyl)cysteamine is a very effective inhibitor of the enwith 10 mM imidazole (pH 8.2) Containing 10 m M L-glutamate and 10 zyme; detailed studies on the kinetics of inactivation by this com-mM MgClz with a flow rate of 60 ml/hr. In each case, at arrow a, pound will be reported later. buffer was changed to include 0.5 M NaCl. At arrow 6, 10

DISCUSSION
These studies show that the specificity of cystamine inhibition of y-glutamylcysteine synthetase is significantly broader than previously appreciated. Earlier work showed that cysteamine and several disulfides closely related in structure to cystamine are not inhibitory (3). However, it is now evident that the enzyme can bind to a cystamine analog that has only one free amino group and also to an analog which is attached to a large structure. The data indicate that enzyme which becomes covalently linked to the matrix attaches by a mechanism involving interaction of an enzyme sulfhydryl group located at or close to the glutamate binding site. Such binding is significantly augmented by ATP. Earlier work showed that prior binding of ATP facilitates the binding of glutamate and of glutamate analogs (4,5,11). It is noteworthy that binding of the enzyme to cystamine-Sepharose is substantially reduced in the presence of glutamate and magnesium ions. Previous studies, which showed that cr-amino-4oxo-5-chloropentanoate (7), and chloroketone analogs of aaminobutyrate (5) are potent inhibitors of the enzyme, also showed that glutamate and magnesium ions protect against such inhibition. Presumably, these inhibitors as well as cystamine bind at, or close to, the glutamate binding site of the enzyme. It is likely that the inhibitors do not bind in exactly the same manner. Thus, whereas ATP increases the interaction with cystamine, this was not observed with the chloroketones.
When the enzyme is applied to cystamine-Sepharose, a large fraction is bound to the column in a form from which the active enzyme may be released by treatment with dithiothreitol. The findings indicate that the enzyme can recognize all or part of the bound cystamine. That the enzyme does not bind to S-(S-methy1)cysteamine-Sepharose suggests that the internal cysteamine-S disulfide moiety of cystamine-Sepharose ( Fig. 4 1 3 is not the recognition site. Since free S-(s-methy1)cysteamine inhibits the enzyme, and free cysteamine does not, it seems probable that the enzyme recognition site of Sepharose-bound cystamine is either the "S"S-CH2-CH2-NH2 moiety or a longer chain of atoms containing this moiety (Fig. 4B). The partitioning of the enzyme on cystamine-Sepharose to yield Sepharose-bound enzyme and unbound reversibly inactivated enzyme might be explained if the initial binding of the enzyme to the column places the reactive enzyme sulfhydryl group in a position in which it can interact with either the internal sulfur atom or with the external sulfur atom of bound cystamine. An alternative possibility is that binding of the enzyme sulfhydryl group to the internal cystamine sulfur atom liberates free cysteamine, which undergoes spontaneous oxidation to form free cystamine, which inhibits some unbound enzyme (Fig. 5).
The studies reported here, which show that a substantially modified cystamine molecule is an inhibitor of y-glutamylcysteine synthetase, suggest that other cystamine analogs may also be effective inhibitors. As discussed elsewhere (3, 12), partial in vivo inhibition of y-glutamylcysteine synthetase may be of therapeutic value in 5-oxoprolinuria (12)(13)(14)(15). Cystamine analogs may thus provide an alternative approach to in vivo inhibition of glutathione synthesis, and be of value in experimental work on the function of glutathione.
Examination of the literature reveals that y-glutamylcysteine synthetase is not unique among enzymes in being potently inhibited by cystamine. Thus, there are published reports indicating that cystamine inhibits at least five other enzymes (phosphorylase phosphatase (16,17), guanylate cyclase (18), indoleamine N-acetyltransferase (19), glycogen synthetase (ZO), and transglutaminase (21)), and that it activates at least two enzymes (fructose diphosphatase (22) and acetyl coenzyme A hydrolase (23)). The published data indicate that the specificity of cystamine in producing these effects varies considerably, but that in most cases, the effects can be reversed by treatment of the enzyme with a thiol. It seems likely that the cystamine-Sepharose matrix described here may therefore be useful as an affinity chromatography procedure for the study of the active sites of other enzymes and possibly also in their purification.