Proc. Natl Acad. Sci. USA Vol. 78, No. 2, pp. 936-939, February 1981 Biochemistry Stimulation of hepatic glutathione formation by administration of L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-prolinase substrate (cysteine delivery system/phosgene/pyroglutamate) JOANNE M. WILLIAMSON AND ALTON MEISTER Department of Biochemistry, Cornell University Medical College, New York, New York 10021 Contributed by Alton Meister, November 12, 1980 ABSTRACT 5-Oxo-L-prolinase, the enzyme that catalyzes the sample of the L isomer which was used in our initial studies. conversion of 5-oxo-L-proline to L-glutamate coupled to the cleav- 5-Oxo-L-[U-'4C]proline and L-[U-'4C]glutamate were obtained age of ATP to ADP and Pi, also acts on L-2-oxothiazolidine-4-car- boxylate (an analog of 5-oxoproline in which the 4-methylene from New England Nuclear. Male mice (NCS strain; 15-25 g) moiety is replaced by sulfur) and ATP to yield cysteine and ADP. were obtained from The Rockefeller University. Pyruvate ki- The enzyme, which exhibits an affinity for the analog similar to nase, lactate dehydrogenase, glutathione reductase, phos- that for the natural substrate, is inhibited by the analog in vitro phoenolpyruvate, NADPH, NADH, glutathione, dithiothrei- and in vivo. L-2-Oxothiazolidine-4-carboxylate thus serves as a tol, ethanolamine, ATP, 5,5'-dithiobis(2-nitrobenzoate) (DTNB), ptent inhibitor of the y-glutamyl cycle at the step of 5-oxopro- and Hepes were obtained from Sigma. 2-Vinylpyridine was ob- linase. Administration of L-2-oxothiazolidine-4-carboxylate to mice that had been depleted of hepatic glutathione led to resto- tained from Aldrich. ration of normal hepatic glutathione levels. Since L-2-oxothiazo- 5-Oxo-L-prolinase has been purified from rat kidney (3); the lidine-4-carboxylate is an excellent substrate of the enzyme, it may enzyme used in the present studies was isolated from rat kidney serve as an intracellular delivery system for cysteine and thus has by a new procedure which led to a final specific activity of 84 potential as a therapeutic agent for conditions in which there is units per mg. Immediately prior to use, the enzyme was freed depletion of hepatic glutathione. of the 5-oxoproline present in the storage buffer by the gel fil- tration procedure described by Penefsky (8). 5-Oxo-L-prolinase catalyzes the ATP-dependent hydrolysis of After incubation, the reaction mixtures (see Fig. 2 and Table 5-oxo-L-proline according to the reaction given in Fig. 1. The 1) were treated with 0.1 vol of 1 M HC1 and placed at 0°C for requirement for energy in this reaction is consistent with the 5 min; an equivalent volume of 1 M Tris was added. Portions position of the equilibrium between 5-oxoproline and gluta- were analyzed for amino acids (glutamate, cystine, S-acetami- mate, which markedly favors the cyclic product (1-3). 5-Oxo- docysteine, and 2,3-diaminopropionic acid) by use of a Durrum prolinase activity has been found in a number of animal tissues model 500 amino acid analyzer. In some experiments, cysteine and has been purified from kidney, a rich source of the enzyme. was determined by reaction with DTNB as described (9). The Earlier work in this laboratory established that 5-oxo-L-proline kinetic values reported in Table 1 were determined by following is a quantitatively significant metabolite of glutathione which ADP formation continuously with the pyruvate kinase/lactate is formed in the 'y-glutamyl cycle by the action of 'y-glutamyl dehydrogenase coupled assay. In these studies, initial velocities cyclotransferase on y-glutamyl amino acids (4). Thus, the en- were determined in reaction mixtures (final volume, 1.0 ml) zyme-catalyzed hydrolysis of5-oxo-L-proline links the reactions containing 100 mM Na Hepes buffer (pH 8.0), 150 mM KC1, involved in the utilization of glutathione (catalyzed by y-glu- 8 mM MgCl2, 2 mM phosphoenolpyruvate, 20 units ofpyruvate tamyl transpeptidase, y-glutamyl cyclotransferase, and cystei- kinase, 20 units of lactate dehydrogenase, 5 mM ATP, 0.3 mM nylglycinase) with those involved in its synthesis (catalyzed by NADH, 2-9 ,ug of 5-oxoprolinase, and 5-200 ,uM substrate or y-glutamylcysteine synthetase and glutathione synthetase). In substrate analog. Activity was followed at 37°C by measuring previous work (5) it was shown that L-2-imidazolidone4-car- the rate of change of absorbance at 340 nm. boxylate, a competitive inhibitor of 5-oxoprolinase, markedly decreases the metabolism of 5-oxoproline in vivo. Here we describe a new heterocyclic substrate of 5-oxopro- RESULTS linase, L-2-oxothiazolidine-4-carboxylate, which is cleaved by When the enzyme was incubated with L-2-oxothiazolidine-4- the enzyme according to the scheme given in Fig. 1. In this carboxylate in the presence of ATP there was rapid formation pathway it is assumed that S-carboxy-cysteine, the initial prod- of ADP and cysteine (Fig. 2, curves 1 and 2). No reaction was uct of hydrolysis, decarboxylates nonenzymatically. We have observed when ATP was omitted (curve 4) or when the substrate found that administration of L-2-oxothiazolidine-4-carboxylate was replaced by the corresponding D isomer (curve 3). That the to mice produces marked inhibition of the metabolism of 5- ratio of ADP to cysteine produced increased with time may oxoproline but not that of glutamate. Administration of this readily be explained by loss ofcysteine due to oxidation. A more compound also stimulates formation of glutathione in the liver. quantitative characterization of the reaction was obtained in studies in which the formation of cysteine was estimated by EXPERIMENTAL PROCEDURES determining derivatives of this amino acid product. The for- Materials. The L and D isomers of 2-oxothiazolidine-4-car- mation of ADP and cysteine (determined as cystine after oxi- boxylate were synthesized by the method of Kaneko et al. (6) dation, or as the corresponding S-acetamido compound after as modified (7). We are indebted to Sidney Weinhouse for a reduction and derivatization) was stoichiometric (Table 1). It is notable that the apparent Km value for the new substrate is The publication costs of this article were defrayed in part by page charge somewhat lower than that found for the natural substrate, 5-oxo- payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: DTNB, 5,5'-dithiobis(2-nitrobenzoate). 936 Downloaded by guest on September 29, 2021 Biochemistry: Williamson and Meister Proc. Natl. Acad. Sci. USA 78 (1981) 937 CH2 -CH2 I I + ATP =m COO- CHCOO- + ADP + Pi .COo- I NH3 H 5-Oxo-L-proline L-Glutamate S CH2 l I + ATP COO- CHCOO- + ADP + Pi I +NH3 L-2-Oxothiazolidine- 4-carboxylate I-C02 HS-CH2 I CHCOO- +NH3 L-Cysteine FIG. 1. Reactions catalyzed by 5-oxo-L-prolinase. L-proline; however, the calculated V.. value for the natural utilization of 5-oxo-L-proline by the purified enzyme; in reac- substrate is higher than that found for the analog. As expected, tion mixtures containing 1 mM 5-oxo-L-proline, addition of L- L-2-oxothiazolidine-4-carboxylate is an excellent inhibitor of the 2-oxothiazolidine4-carboxylate at concentration of 1, 5, and 10 mM produced 73, 92, and 97% inhibition of glutamate for- mation, respectively. Under these conditions, D-2-oxothiazo- lidine-4-carboxylate did not inhibit. The data in Fig. 3 show that L-2-oxothiazolidine-4-carboxy- 1.5 Table 1. Action of 5-oxo-L-prolinase on L-2-oxothiazolidine-4-carboxylate Products formed, 1.0 nmol :._ Amol/ ae Substrate Amino App. K., min/ 0 ADP* acid ID Compound mM AM mg cil 5-Oxo-L-proline 2 924 916t 5 1.34 Q 0.5 L-2-Oxothiazolidine- 2 478 466t 2 0.73 4-carboxylate 499 463§ D-2-Oxothiazolidine- 4-carboxylate 5 96 0t1 - - L-2-Imidazolidone- 4-carboxylate 2 930 oil 14 1.45 The reaction mixtures contained (final volume, 0.5 ml) 100 mM Na Hepes buffer (pH 8.0), 150 mM KCl, 8 mM MgCl2, 2 mM phosphoen- 0 10 20 30 40 olpyruvate, 5 mM ATP, 10 units of pyruvate kinase, 20 ,tg of 5-oxo- Time, min prolinase, and substrate as indicated; incubation was for 30 min at 370C. FIG. 2. Cleavage of L-2-oxothiazolidine-4-carboxylate by 5-oxo-L- * Determined by use of pyruvate kinase/lactate dehydrogenase cou- prolinase. The reaction mixtures (final volume, 1.5 ml) contained 100 pled assay; all values were corrected by subtracting a blank value mM Na Hepes buffer (pH 8.0), 150 mM KCI, 8mM MgCl2, 2mM phos- of 54 nmol. phoenolpyruvate, 10 units of pyruvate kinase, 100 ,ug of 5-oxoproli- t Determined as glutamate. nase, and: in curves 1 and 2, 5 mM L-2-oxothiazolidine-4-carboxylate t Determined after reduction with KBH4 and reaction with iodoace- and 5 mM ATP; in curve 3, 5 mM D-2-oxothiazolidine-4-carboxylate tamide (10). and 5 mM ATP; in curve 4,5 mM L-2-oxothiazolidine-4-carboxylate but § Determined after conversion to cystine by oxidation. ATP was omitted. At the indicated intervals, portions were analyzed <20 nmol. for ADP and for cysteine by reaction with DTNB (9). II <5 nmol. Downloaded by guest on September 29, 2021 938 Biochemistry: Williamson and Meister Proc. Nad Acad. Sci.'USA 78 (1981) 2 u, ._I:Io -6-0 020 bo BE X if l "~~~~~20~~~~~~ 6040 B- 60-40 Time after injection, hr 20 9 , , [ , FIG. 4. Increase in hepatic levels of glutathione (GSH) after in- jection of L-2-oxothiazolidine-4-carboxylate.