interaction of the d-isomer of gamma-methylene glutamate with an

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 36, Issue of December 25, pp. 17134-17137,1986 Printed in U.S.A.

Interaction of the D-Isomer of ?-Methylene Glutamate with an Active Site Thiol of y-Glutamylcysteine Synthetase*

(Received for publication, June 20, 1986)

Royce P. Simondsen and Alton Meister From the Department of Biochemistry, Cornell University Medical College, New York, New York 10021

7-Glutamylcysteine synthetase has a thiol group in the vicinity of its glutamate-binding site. During ef- forts to find a covalently bound inhibitor, interaction of the enzyme with y-methylene glutamate was exam- ined because this analog of glutamate, which has an a,@-unsaturated moiety, would be expected to bind at the glutamate site and might react with an active site thiol. y-Methylene glutamate, which is not a significant substrate, inhibits the enzyme competitively toward glutamate. Preincubation of the enzyme with y-methylene DL-glutamate led to substantial inactivation which was dependent upon the presence of Mg2+ or Mn2+; glutamate protected against inactivation. Inac- tivation was observed with the D-isomer of y-methylene glutamate, but not with the corresponding L-isomer. The inactivated enzyme contains close to 1 mol of y-methylene glutamate/mol of enzyme. Studies in which enzyme inactivated by treatment with [14C]y- methylene glutamate was hydrolyzed indicate that y- methylene glutamate reacts with an active site thiol.

y-Glutamylcysteine synthetase, the enzyme that catalyzes the first step in the synthesis of glutathione (reaction I), has a thiol group

L-Glutamate + L-cysteine + ATP (1)

in the vicinity of the glutamate-binding site (1). This thiol, which does not react with 5,5‘-dithiobis(nitrobenzoate) (l), readily interacts with cystamine to form a mixed disulfide (2- 4); this produces inactivation, which can be prevented by glutamate and reversed by treatment with dithiothreitol (2, 3). Glutamate also protects the enzyme against inactivation by ~-2-amino-4-oxo-5-chloropentanoate (5 , 6) and by the L- and D-isomers of 3-amino-1-chloro-2-pentanone (3). When the enzyme is incubated with cystamine and then with one of these chloroketones followed by treatment with dithiothreitol, there is no loss of activity. These observations indicate that the active site thiol can react with cystamine and with the chloroketones. There is indirect evidence that y-glutamyl phosphate is formed as an enzyme-bound intermediate in reaction 1 (7, 8). Methionine sulfoximine (9) (and other sulfoximines such as prothionine sulfoximine and buthionine sulfoximine (10-12)) inactivates the enzyme in the presence

*This work was supported in part by a grant from the United States Public Health Service, National Institutes of Health (AM 12034). A preliminary account of this research has appeared (Simondson, R. P., and Meister, A. (1986) Fed. Proc. 46, 313). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

s L-y-glutamyl-L-cysteine + ADP + Pi

of ATP. These inactivators are phosphorylated on the sulfoximine nitrogen atom; the amino acid sulfoximine phosphates bind tightly but noncovalently to the enzyme, thus inactivat- ing it. Such phosphorylation appears analogous to the formation of y-glutamyl phosphate in the normal catalytic reaction. Treatment of the enzyme with cystamine prevents its interaction with methionine sulfoximine (3).

To elucidate further the mechanism of action of the enzyme, we have investigated its interaction with y-methylene glutamate, an amino acid found (as the dicarboxylate and as the corresponding w-amide) in certain plants (13,14), and known to interact with several enzymes that act on glutamate (see, for example Refs. 15-18). This glutamate analog would be expected to bind at the glutamate-binding site of y-glutamylcysteine synthetase, and might also react with the active site thiol through a Michael-type addition reaction:

+ RSH + -0OC-CHNH3-CH-C-COO-

I1 CHz

(2) +- + -OOC-CHNH&HZ-CH-COO-

I CHzSR

Powell et al. (19) prepared the thioethers formed by reaction of y-methylene glutamic acid with cysteine, glutathione, and cysteamine. They did not find such adducts in germinating peanuts, which contain y-methylene glutamate as well as these thiols. These investigators suggested that y-methylene glutamate might inactivate enzymes that contain active site thiols.The present studies show that y-glutamylcysteine synthetase is inactivated by y-methylene glutamate and that inactivation involves reaction of this compound with an active site thiol. It is notable that the D-isomer of y-methylene glutamate (but not the L-isomer) inactivates the enzyme.

EXPERIMENTAL PROCEDURES’

RESULTS

Inhibition of y-Glutamylcysteine Synthetase by y-Methylene Glutamate-y-Methylene L-glutamate and y-methylene-L-

Portions of this paper (including “Experimental Procedures” and Figs. 1, 4, 5, and 6) are presented in miniprint at the end of this paper. The abbreviation used is: HEPES, 4-(2-hydroxyethyl)-l-pi- perazineethanesulfonic acid. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86 “2066, cite the authors, and include a check or money order for $2.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

17134

y-Methylene Glutamate and y-Glutamylcysteine Synthetase 17135

glutamine were tested (in place of L-glutamate) as substrates of y-glutamylcysteine synthetase in the standard assay containing enzyme (10 units); incubation was carried out for 6 h. Neither compound was converted to a dipeptide as determined by amino acid analysis. y-Methylene L-glutamate was found to be a competitive inhibitor of the enzyme (toward L-glutamate) with an apparent K, of 2.5 mM, whereas the apparent Ki for the racemic mixture was 4.5 mM (Fig. 1, A and E , Miniprint). The apparent K,,, for t-glutamate was 1.6 mM; previously values of 1.8 (27) and 1.6 were observed (28). The apparent affinity of y-methylene L-glutamate for the enzyme is thus somewhat lower than that of L-glutamate.

Inactivation of y-Glutamylcysteine Synthetase by y-Meth- ylene DL-Glutamate-when the enzyme was incubated with y-methylene DL-glutamate in the presence of M$+ or Mn2+ and then assayed after 100-fold dilution into the assay mixture, there was a marked loss of activity. Inactivation was not observed in the absence of M$+ or Mn2+ and was greater with Mn2+ than with M$+ (Fig. 2).

Fig. 3 illustrates the behavior of the enzyme toward the racemic mixture of y-methylene glutamate and the separated L- and D-isomers of this compound in the presence of Mn2+. Inactivation was rapid with the racemic mixture (Curue 2) and with y-methylene D-glutamate (Curues 3 and 4 ) . The L- isomer did not inactivate (Curue 1 ). The L-isomer appears to protect against inactivation, since the racemic mixture inactivated less rapidly than did the D-isomer. The inactivation

100 0-"0--.-0--.

!- J

l o t i I I I I I

1.0 2.0 3.0 4.0 5.0 1 I [Mg',+] (mMII I

0.5 1 .o 1.5 2 .o [Mn2+] (mM)

FIG. 2. Inactivation of y-glutamylcysteine synthetase by y- methylene DL-ghtamate. Enzyme (10 units) was incubated at 37 "C in a solution (final volume, 0.1 ml) containing Tris/HCl buffer (50 mM, pH 8.2) for 10 min prior to addition of MgCI, or MnC1, (final concentrations indicated) and y-methylene DL-glUtamate (10 mM). Incubation was continued for 30 min, at which time a portion (10 pl) was removed and assayed for ADP formation by the coupled enzyme assay. Open circles, Mn2+ controls; closed circles, M e controls; squares, M e plus y-methylene DL-glutamate; triangles, Mn2+ plus y-methylene DL-glutamate.

2 6 IO 14 18 22 26 30 60 90 PREINCUBATION PERIOD (minutes)

FIG. 3. Inactivation of y-glutamylcysteine synthetase by y- methylene D-glutamate. The enzyme (10 units) was incubated at 37 "C in a solution (final volume, 0.1 ml) containing Tris/HCl buffer (50 mM, pH 8.2). MnC1, (0.25 mM), and inhibitor. Curve 1, y- methylene L-glutamate (10 mM); Curve 2, y-methylene DL-glutamate (10 mM); Curve 3, y-methylene D-glutamate (4.5 mM); Curve 4, y- methylene D-glutamate (10.8 mM). Portions (10 pl) were removed at the indicated times and assayed for ADP formation.

TABLE I Interaction of the enzyme with y-methylene glutamate

The reaction mixtures (final volume, 0.1 ml) contained Tris/HCl buffer (pH 8.2,50 mM), M&l, (5 mM), enzyme (10 units), and added compounds. The solutions, prior to mixing, were placed at 37 "C for 10 min. Cystamine (0.2 mM), y-methylene DL-glutamate (yMG) (20 mM), L-glutamate (10 mM), and L-a-aminobutyrate (10 mM) were added as indicated. In experiment 3, cystamine was added 5 min before adding yMG. In experiments 5 and 6, yMG was added 10 min after adding glutamate and a-aminobutyrate, respectively. After incubation of the complete reaction mixtures for 30 min at 37 "C, they were assayed for enzyme activity (after 100-fold dilution) in the presence and absence of 1 mM dithiothreitol.

% Activity remaining

added Without With

1 None 100 93.2

Experiment Compounds

dithiothreitol dithiothreitol

2 Cystamine 6.8 106 3 Cystamine + yMG 6.0 112 4 yMG 51.6 5 Glutamate + yMG

53.6 76.0 76.0

6 a-Aminobutyrate + yMG 57.7 59.3

rate, determined in experiments with three concentrations of y-methylene D-glutamate, was found to be 0.0214 min" mM" (Fig. 4, Miniprint). This rate did not show saturation behavior up to a concentration of 10 mM inhibitor.

That the inhibitor binds to the enzyme at the glutamate binding site is consistent with data given in Table I. As discussed above, inactivation by cystamine may be reversed by inclusion of dithiothreitol in the assay mixture (3). Similar results were found in experiments 2 and 3 (in the presence and absence of y-methylene glutamate). Glutamate partially protects against inactivation, whereas L-a-aminobutyrate does not (experiments 4-6).

To characterize further the inactivation, the enzyme was treated with y-methylene DL-[2-'4C]gl~tamate and then subjected to gel filtration. The elution profile of the reaction

17136 y-Methylene Glutamate and y-Glutamylcysteine Synthetase

mixture from Sephadex G-50 is shown in Fig. 5 (Miniprint). The enzyme eluted in this experiment was 87% inactivated and was found to bind 0.90 mol of y-methylene [2-'4C]glutamate/mol of enzyme. In a duplicate experiment, 0.82 mol of y-methylene [2-'4C]glutamate was bound per mole of enzyme that was 89% inactivated. These experiments indicate binding of close to 1 mol of y-methylene [2-'4C]glutamate/mol of inactivated enzyme.

Enzyme that was 87% inactivated by treatment with y- methylene [2-14C]glutamate and isolated by gel filtration as described above was subjected to acid hydrolysis (6 M HC1, 110 "C, 48 h), and the hydrolysate was chromatographed on a Durrum amino acid analyzer modified for collection of samples; the radioactivity of the samples was determined (Fig. 6A, Miniprint). More than 90% of the applied radioactivity was recovered in the effluent. The first peak to emerge (void volume) contains unhydrolyzed peptide material. Of the radioactivity present in fraction 5-23 (295 dpm), 45% (132 dpm) was found in fractions 20 and 21, which eluted in the position of an authentic sample of the adduct formed between cysteine and y-methylene glutamate. Fractions 6, 9, 10, and 11 also contained radioactivity, the chemical nature of which has not been determined. In a comparable study in which hydrolysis was carried out enzymatically (Fig. 6B) (see "Experimental Procedures"), about 240 dpm were found in fractions 5-21, and fractions 19 and 20 (corresponding to the adduct) contained 153 dpm or 64% of the total. Much less radioactivity was found in fractions 9-12 in this hydrolysate, suggesting that the radioactivity found in this region of the chromato- gram of the acid hydrolysate represents material produced by chemical degradation. Consistent with these interpretations are the findings that mild acid hydrolysis (4 M HC1, 100 "C, 6.5 h) of the enzymatically hydrolyzed material led to a 20% decrease in the radioactivity found in fractions 3-5 with concomitant increases in the radioactivity found in fractions 7-11; there was also a 7% decrease in the radioactivity found in the peak corresponding to the adduct.

The findings are consistent with the conclusion that the

FIG. 7. Stereophotographs of models of the D (left) and L (right) isomers of y-methylene glutamate in the extended conformation. The model of the D-isomer is rotated (below) as discussed in the text. (The amino hydrogen atoms are omitted).

major amino acid residue of the enzyme involved in inactivation by y-methylene glutamate is cysteine. That the recov- ery of adduct was less than 100% may probably be ascribed to degradation of the adduct during hydrolysis and to me- chanical losses. Treatment of the authentic adduct with 6 M HC1 at 110 "C for 48 h led to little loss, but when this treatment was carried out in the presence of added denatured enzyme there was a loss of about 20%. Although the findings strongly suggest that y-methylene glutamate interacts with a cysteine moiety, we cannot completely exclude the possibility that this amino acid analog also interacts to a smaller extent with other amino acid side chains of the enzyme.

DISCUSSION

The previous studies on this enzyme indicate that it has a thiol group which may react with several chloroketones or with cystamine. These interactions, which are inhibited by L- glutamate, lead to an inactivated enzyme. This enzyme thiol moiety might be involved in catalysis, for example, by reacting with y-glutamyl phosphate to form a y-glutamyl-S-enzyme intermediate. However, the fidings could also be explained if reaction of an enzyme thiol group with cystamine or chloroketones simply blocked the binding site for glutamate.

The structural analogy between y-methylene glutamate and glutamate, and the findings reported here, suggest that both compounds bind to the same enzyme site. It is relevant to note that the enzyme interacts with both L-glutamate and D- glutamate (28). The binding of D-glutamate to this active site may be explained in a manner similar to that previously deduced for the binding of the L- and D-isomers of glutamate to glutamine synthetase (29-31). In this interpretation, L- glutamate binds to the enzyme in an extended conformation in which the carboxyl groups are as far apart as possible, or almost so. (Neither glutamine synthetase nor y-glutamylcysteine synthetase interacts significantly with aspartate.) The D-glutamate molecule can attach to the same enzyme sites that bind the L-glutamate molecule if the extended form of D-glutamate is rotated 69" about an axis through carbon atoms 1, 3, and 5. If one carries out this manipulation with models of the L- and D-isomers of y-methylene glutamate, one can achieve a rotated position of y-methylene D-glutamate in which the a-carboxyl carbon atom, the y-carboxyl carbon atom, and the a-amino nitrogen are in virtually the same positions as those of the corresponding L-isomer. In- spection of these models reveals that the position of the y- methylene moiety is markedly different in the two isomers (Fig. 7). Since only the D-isomer interacts with the enzyme to form a covalent linkage, it may be reasonably proposed that the y-methylene moiety of y-methylene D-glutamate is close to the position of the active site thiol, whereas that of the L- isomer is not. These considerations and the experimental observations reported here suggest that the enzyme thiol group is oriented toward one side of the enzyme-bound glutamate molecule and that it is in the proximity of the y- carboxyl group. Thus, it is possible that the enzyme thiol might form a y-glutamyl-S-enzyme linkage. This intriguing possibility remains to be explored further.

Recent studies on glutathione metabolism, transport, and function (32) have been facilitated by use of the sulfoximine inhibitors of y-glutamylcysteine synthetase, for example, buthionine sulfoximine (11). Administration of such inhibitors leads to substantial depletion of cellular glutathione. The present studies suggest that y-methylene glutamate might also be useful for decreasing cellular glutathione levels.

Acknowledgments-We thank Dr. Francesco Infante for skillful

y-Methylene Glutamate and y-Glutamylcysteine Synthetase 17137

assistance in the studies on the amino acid analyzer and Dr. William Moore for helpful discussions. We are particularly indebted to Dr. Gary K. Powell and Dr. Eugene E. Dekker for providing us with samples of y-methylene L-glutamate and y-methylene DL-['*C]glutamate.

REFERENCES

2. Griffith, 0. W., Larsson, A., and Meister. A. (1977) Biochem. Biophys. Res. 1. Seelig, G. F., and Meister, A. (1984) J. Biol. Chem. 269,3534-3538

3. Beamer, R. L., Griffith, 0. W., Gass, J. D., Anderson, M. E., and Meister,

4. Lebo, R. V., and Kredich, N. M. (1978) J. Biol. Chem. 253,2615-2623 5. Khedouri, E., Anderson, P. M., and Meister, A. (1966) Biochemistry 6,

6. Sekura, R., and Meister, A. (1977) J. BioL Chem. 252,2606-2610 7. Orlowski, M., and Meister, A. (1971) J. Biol. Chem. 246, 7095-7105 8. Meister, A. (1974) in The Enzymes (Boyer, P. D., ed) Vol. 10,3rd. Ed, pp.

9. Richman, P. G., Orlowski, M., and Meister, A. (1973) J. Biol. Chem. 248,

10. Griffith, 0. W., Anderson, M. E., and Meister, A. (1979) J. Biol. Chem.

11. Griffith, 0. W., and Meister, A. (1979) J. Biol. Chern. 254,7558-7560 12. Griffith, 0. W. (1982) J. Biol. Chem. 267,13704-13712

Commun. 79,919-925

A. (1980) J. Biol. Chem. 265, 11732-11736

3552-3557

671-697, Academic Press, New York

6684-6690

264,1205-1210

13. 14.

15. 16. 17. 18.

19.

20. 21. 22. 23.

24. 25.

26.

27.

28.

30. 29.

31. 32.

Done, J., and Fowden, L. (1952) Biochem. J. 51,451-458 Zacharius. R. M.. Pollard. J. K.. and Steward, F. C. (1954) J. Am. Chern.

Meister, A. (1954) J. Biol. Chem. 210,17-35 Hornby, D. P., and En el P C (1983) Int. J. Biochem. 16,495-500 Homola, A. D., and Defkkr, E. E. (1967) Biochemistry 6, 2626-2634, Marcus. A,. Feelev, J., and Shannon, L. M. (1963) Arch. Biochem. Btophys.

Soc. 76', 1961-'1962

- . . 100,.80-85

Res. Comrnun. 106, 1361-1367 Powell, G. K., Winter, H. C., and Dekker, E. E. (1982) Biochem. Biophys.

Seeli G F and Meister, A. (1985) Methods Enzymol. 113,379-390 Pow& G. K., and Dekker, E. E. (1981) Prep. Biochem. 11,339-350 Wailes, P., Whiting M. C., and Fowden, L. (1954) Nature 174,130-131 Benson, J. V., Gordon, M. J., and Patterson, J. A. (1967) Anal. Biochem.

Chin, C. C. Q., and Wold, F. (1974) Anal. Biochem. 61,371-391 Seelig, G. F., Simondsen, R. P., and Meister, A. (1984) J. Biol. Chern. 269,

IS, 228-240

9!?65-Q267 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J .

Sekura, R., Hochreiter, M., and Meister, A. (1976) J. Biol. Chem. 261,

Orlowski, M., and Meister, A. (1971) Biochemistry 10,372-380 Meister, A. (1968) Adu. Enzymol. 31,183-218 Meister, A. (1974) in The Enz mes (Boyer, P. D., ed) Vol. 10, 3rd. Ed, pp.

Gass, J. D., and Meister, A. (1970) Biochemistry 9,1380-1390 Meister, A., and Anderson, M. E. (1983) Annu. Reu. Biochem. 52, 711-760

""_ "_. Biol. Chern. 193,265-275

2263-2270

699-754, Academic Press, d w York

interaction of the d-isomer of gamma-methylene glutamate with an

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