Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2464-2468, April 1988 Biochemistry On the active site thiol of y-glutamylcysteine synthetase: Relationships to catalysis, inhibition, and regulation (glutathione/cystamine/Escherichia coli/kidney/enzyme inactivation) CHIN-SHIou HUANG, WILLIAM R. MOORE, AND ALTON MEISTER Cornell University Medical College, Department of Biochemistry, 1300 York Avenue, New York, NY 10021 Contributed by Alton Meister, December 4, 1987 ABSTRACT y-Glutamylcysteine synthetase (glutamate- dithiothreitol, suggesting that cystamine forms a mixed cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia disulfide between cysteamine and an enzyme thiol (15). coli strain enriched in the gene for this enzyme by recombinant Inactivation of the enzyme by the L- and D-isomers of DNA techniques. The purified enzyme has a specific activity of 3-amino-1-chloro-2-pentanone, as well as that by cystamine, 1860 units/mg and a molecular weight of 56,000. Comparison is prevented by L-glutamate (14). Treatment of the enzyme of the E. coli enzyme with the well-characterized rat kidney with cystamine prevents its interaction with the sulfoxi- enzyme showed that these enzymes have similar catalytic prop- mines. Titration of the enzyme with 5,5'-dithiobis(2- erties (apparent Km values, substrate specificities, turnover nitrobenzoate) reveals that the enzyme has a single exposed numbers). Both enzymes are feedback-inhibited by glutathione thiol that reacts with this reagent without affecting activity but not by y-glutamyl-a-aminobutyrylglycine; the data indicate (16). 5,5'-Dithiobis(2-nitrobenzoate) does not interact with that glutathione binds not only at the glutamate binding site but the thiol that reacts with cystamine. Evidence has also been also at a second site on the enzyme that interacts with the thiol obtained that y-methylene-D-glutamate, which inactivates moiety of glutathione but not with a methyl group. Both the enzyme, binds in the glutamate binding site of the active enzymes are inactivated by buthionine sulfoximine in the pres- center and forms a covalent linkage with the active site thiol ence of ATP, suggesting a common y-glutamyl phosphate through a Michael-type addition reaction (17). The enzyme is intermediate. However, unlike the rat kidney enzyme that has also inactivated by S-sulfocysteine and S-sulfohomocys- an active center thiol, the bacterial enzyme is insensitive to teine; these inactivators are tightly but noncovalently bound cystamine, r-methylene glutamate, and S-sulfo amino acids, and their interaction with the enzyme is postulated to indicating that it does not have an active site thiol. Thus, the rat involve effects of the active site thiol (18). These and earlier kidney and E. coli enzymes share several catalytic features but (14) observations have led to the suggestion that the active differ in active site structure. If the active site thiol of the rat center thiol may play a role in the mechanism of action of is in this enzyme. kidney enzyme involved catalysis, which seems likely, there In contrast to the results summarized above obtained in would appear to be differences in the mechanisms of action of studies on rat kidney y-glutamylcysteine synthetase, the the two y-glutamylcysteine synthetases. findings on the y-glutamylcysteine synthetase ofEscherichia coli, reported here, indicate that the bacterial enzyme has The first step in the synthesis of glutathione is catalyzed by properties that do not reflect the presence ofan active center y-glutamylcysteine synthetase (glutamate-cysteine ligase; thiol. Nevertheless, the bacterial enzyme exhibits a number EC 6.3.2.2) [Reaction 1 (ref. 1, pp. 671-697)]. This reaction, of characteristics that closely resemble those of the mam- usually the rate-limiting step in glutathione synthesis, is malian enzyme, including, for example, high turnover num- feedback-inhibited by glutathione (2). y-Glutamylcysteine ber, feedback inhibition by glutathione, and inactivation by synthetase, like glutamine synthetase (ref. 1, pp 699-754, incubation with buthionine sulfoximine in the presence of and refs. 3 and 4), is inactivated by methionine sulfoximine ATP. A preliminary account of this work has been presented in the presence of ATP (5). Methionine sulfoximine and (*; ref. 19). certain other sulfoximines, such as buthionine sulfoximine [which inhibits y-glutamylcysteine synthetase but not gluta- EXPERIMENTAL PROCEDURES mine synthetase (6-9)], are phosphorylated by ATP on the enzymes; the phosphorylated sulfoximines bind tightly, but Materials. Agarose-hexane-ATP (Al6; type 2), CM-Seph- noncovalently, to the enzymes, thus producing inhibition. adex C-50, Sephadex G-100, and Sephadex G-150 were purchased from Pharmacia. DE-52 was obtained from What- L-Glutamate + ATP + L-cysteine man. L-Buthionine-(SR)-sulfoximine (6-8), S-sulfocysteine L-y-glutamyl-L-cysteine + ADP + Pi [1] (18), S-sulfohomocysteine (18), and y-methylene glutamate (17) were synthesized as described. Other compounds were The presence of a thiol at the active site of y-glutamyl- obtained from Sigma. cysteine synthetase was suggested by the finding (10) that Microorganisms. Previously a strain of E. coli enriched by highly purified 'y-glutamylcysteine synthetase from rat kid- recombinant DNA techniques in its content of the two ney is inactivated by low concentrations of L-2-amino-4-oxo- synthetases required for glutathione synthesis was described 5-chloropentanoate (11), that such inactivation is associated (20, 21). The genes for the synthetases were isolated and with covalent binding of the inactivator, and that glutamate inserted into a vector that was used to transform the original protects against inactivation. Subsequently it was found that strain. The transformed strain, in an immobilized form, has the enzyme is strongly inactivated by cystamine (12-14) and been used for the synthesis of isotopically labeled gluta- that inactivation by cystamine is reversed by treatment with thione (22) and for the synthesis of glutathione analogs (23). In the present work a bacterial strain enriched in its content The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" *Huang, C.-S., Moore, W. R. & Meister, A., American Society of in accordance with 18 U.S.C. §1734 Alely to indicate this fact. Biological Chemists Meeting, June 6-10, 1987, Philadelphia, PA. 2464 Downloaded by guest on October 4, 2021 OXYGEN COST AND EFFICIENCY IN HEALTH AND EMPHYSEMA 497 z 0 Z 0 - 7s I/ . / II100 200. O 0 0E NO1/ d)/ P1 80 10V CLJ 0 2 150 x 4p,, x > -a Zn 60 - C) u0 -~ 100 -w°X 1. r0 40 IZ I z I . ol 0~. :-0c LA 20 0 10 20 30 4I0 0 6 3 'D VENTILATION A z (L/min. B.T.P.S.) 0 2.0 4.0 6.0 8.0 FIG. 3. THE CHANGE IN OXYGEN CONSUMPTION As- ADDED MECHANICAL WORK SOCIATED WITH CHANGE IN MINUTE VENTILATION IN 22 (kgm.m./min.) PATIENTS WITH EMPHYSEMA FIG. 4. THE EXTRA OXYGEN CONSUMPTION REQUIRED Also shown are isopleths representing the oxygen TO HANDLE ADDED RESPIRATORY WORK LOADS IN NINE cost of per liter of ventilation. breathing NORMAL AND SEVEN EMPHYSEMATOUS SUBJECTS Also shown are isopleths representing the efficiency of tion of the respiratory muscles per liter of ventila- the respiratory muscles for handling added work loads. tion was considerably higher in the patients with 0 represents normal subjects; X represents emphysema- emphysema than in the normal subjects. The tous subjects. cost ranged between 0.45 to 1.87 ml. per L. of ventilation (mean 1.16) in the normal subjects, was high for even slight increases in ventilation while it ranged between 1.68 and 18.50 ml. per L. and rose still further when ventilation was in- of ventilation (mean 5.96) in the emphysematous creased. subjects. The extra oxygen consumption associated with In Figures 2 and 3 the increments in oxygen added work loads in nine normal subjects and consumption associated with changes in ventilation seven patients with emphysema is shown in Fig- in the normal subjects and patients with emphy- ure 4. It can be seen that the extra oxygen sema are shown. It can be seen that in the normal consumption per unit added work load was greater subjects the oxygen cost of an increase in ventila- in the patients with emphysema than in the tion of 30 L. per minute above the resting ventila- normals. tion was less than 2 ml. per L. On the other hand, In Table III the calculated efficiencies of the in the subjects with emphysema the oxygen cost respiratory muscles of the nine normal and seven TABLE III Total mechanical work and efficiency of the respiratory muscles in normal and emphysematous subjects Normal Emphysematous Total mechanical work Total mechanical work Subj. Efficiency at rest Subj. Efficiency at rest % Kg. M./L. Kg. M./min. % Kg. M./L. Kg. M./min. 1 10.80 0.173 1.52 17 2.74 0.141 1.39 2 7.45 0.172 1.38 18 1.45 0.203 1.77 3 7.60 0.123 0.82 19 2.30 0.159 1.39 4 8.20 0.280 2.69 20 2.04 0.120 0.83 5 11.10 0.326 3.00 21 1.24 0.188 1.20 6 8.04 0.204 2.74 22 1.47 0.284 2.68 7 7.20 0.203 1.83 23 1.60 0.123 1.22 8 7.43 0.215 1.89 9 9.35 0.349 2.27 Mean 8.58 0.227 2.02 1.83 0.174 1.49 Downloaded by guest on October 4, 2021 2466 Biochemistry: Huang et al. Proc. Natl. Acad. Sci. USA 85 (1988) 1.0, and 0.3 mM. The relative rates of reaction found with Table 2.