Enzymatic catalysis of the reversible sulfitolysis of glutathione disulfide and the biological reduction of thiosulfate esters

ARCHIVES

OF BIOCHEMISTRY

Enzymatic Disulfide BENGT

AND

163, 283-289 (19’ih)

BIOPHYSICS

Catalysis

of the Reversible

and the Biological MANNERVIK,

Department

Reduction

GUN PERSSON,

of Biochemistry,

Sulfitolysis

Uniuer.Gty

of Thiosulfate

AND

of Stockholm,

Received January

of Glutathione

STELLAN

Esters

ERIKSSON

Stockholm,

Sweden

2, 1974

Rat liver supernatants were shown to contain an enzymatic activity catalyzing in both forward and reverse directions the reversible sulfitolysis of glutathione disulfide. The enzymatic sulfitolysis has maximal activity at pH 7. S-Sulfoglutathione, which is a product of the sulfitolysis, was isolated by passage through an ion-exchange column. Three different assays were applied to determine S-sulfoglutathione, viz., methods based on the ninhydrin reaction, the formation of a thiazoline derivative in strong acid, and the use of radioactively labeled glutathione. The reversal of the sulfitolysis, i.e., the reaction of S-sulfoglutathione with glutathione, was studied directly by determination of sulfite with radioactive N-ethylmaleimide, or indirectly by coupling to the NADPH- and glutathione reductaselinked reduction of glutathione disulfide. Chromatographic analysis of rat liver supernatants demonstrated that all fractions catalyzing the reversible sulfitolysis did also catalyze the previously studied thiol-disulfide interchange of glutathione and the mixed disulfide of cysteine and glutathione. The reduction of thiosulfate esters, such as S-sulfocysteine and trimethylammoniumethylthiosulfate, with glutathione was also catalyzed by the enzyme active in the sulfitolysis, which indicates an important biosynthetic role of the enzyme in microorganisms synthesizing cysteine via S-sulfocysteine. The enzyme is also capable of participating in the formation of the naturally occurring S-sulfoglutathione.

The reaction of S-sulfoglutathione (GSSO,H)’ with glutathione (GSH): GSSO,-

+ GSH = GSSG + HSO,-

has been observed to be catalyzed by a partially purified thioltransferase from rat liver (1). However, the enzymatic nature of this catalysis was not clearly established. The reversal of this reaction, i.e., the sulfitolysis of GSSG, has not previously been subject to investigations concerning the possibility of catalysis in uiuo, due to the fact that the spontaneous reaction is fairly rapid. The present paper demonstrates that the reaction is catalyzed in both directions by rat liver supernatants and gives evidence for the conclusion that ’ Nonstandard abbreviations: CySSG, the mixed disulfide of cysteine and glutathione; CySS03H, Ssulfocysteine; GSSO,H, S-sulfoglutathione; and NEM, N-ethylmaleimide. 283 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

the catalysis is enzymatic. The results also indicate that the activity is linked to the enzyme previously studied, which catalyzes thiol-disulfide interchange reactions of low molecular-weight compounds (2). Furthermore, other thiosulfate esters can be used as substrates by the same enzyme. A preliminary report of a part of this investigation has been presented (3). MATERIALS

AND

METHODS

Chemicals The following chemicals were obtained from the suppliers indicated and were used without further purification: GSH, NADPH, and yeast glutathione reductase (Sigma); GSSG (Boehringer): CM-cellulose CM 32 (Whatman); Sephadex G-25 Fine (Pharmacia); [35S]GSH (Schwarz); [“CINEM (NEN Chemicals); 2,5-diphenyloxazole and 1,4-bis-2.(4 methy&phenyloxazolyl)-benzene (Packard); and human serum albumin (KABI).

284

MANNERVIK.

PERSSON.

GSSO,H and the mixed disulfide of cysteine and glutathione (CySSG) were prepared according to previously published procedures of this laboratory (4, 5). S-Sulfocysteine (CySSO,H) was synthesized according to Segel and Johnson (6) and trimethylammoniumethylthiosulfate according to Klayman and Gilmore (7). [YS]GSSG was prepared by aeration of an aqueous solution of [“S ]GSH (pH 7). The purity of the synthesized compounds was checked by paper electrophoresis (cf. Refs. 4 and 5).

Rat Liver Supernatants Livers from male Sprague-Dawley rats were homogenized in 0.25 M sucrose and the homogenate was diluted with sucrose to a concentration of 20% (w/v) and centrifuged. The supematant obtained after centrifugation for 60 min at 105,OOOg was used as the enzyme source. Before column chromatography the supernatant was passed through a Sephadex G-25 Fine column equilibrated with 10 mM sodium phosphate, pH 6.0, containing 1 mM EDTA. The bed volume was five times the sample volume.

Assays of the Sulfitolysis

of GSSG

The formation of GSSO,H was used as a measure of the sulfitolysis of GSSG. GSSO,H was separated from other glutathione derivatives, and was normally determined with the quantitative ninhydrin analysis (method A) or with the radiometric method (method B). In some experiments GSSO,H liberated in the reaction was determined in the form of a thiazoline derivative (method C, cf. Ref. 8). Method A. The sulfitolysis was carried out at 30” C in a reaction system of 1 ml of 40 mM sodium phosphate buffer (pH 7.0) containing0.4 mM GSSG, 1 mM Na,SO, , and enzyme. The reaction was started by addition of sulfite and was stopped with 5 ~1 of concentrated H,SO,. The reaction mixture was then applied to a 0.5 x 4-cm Dowex 50 W (H’) column (X8, 200-400 mesh), washed into the bed with 0.5 ml, and eluted with a l-ml portion of deionized water. GSSO,H is the only glutathione derivative present, which is eluted under these conditions. The effluent (2.5 ml) was neutralized with 50 ~1 of 2 M NaOH, and a l-ml sample was then mixed with 2 ml of ninhydrin reagent and boiled for 20 min on a water bath. The ninhydrin reagent consisted of 25 ml of 3% ninhydrin dissolved in methoxyethanol + 0.1 ml of SnCl, (stock solution: 4.51 g of SnC1,.2H,O + 1.7 ml coned HCl, diluted to 10 ml with water) + 25 ml of 0.2 M sodium citrate pH 5.70. The reagent is stable for ca. 2 hr after mixing of the components. The boiled samples were cooled in water and diluted with 5 ml of 50% ethanol, after which the absorption at 570 nm was determined (cf. Ref. 9). Method B. The reaction system contained in a final volume of 0.5 ml: 0.5 mM [‘YS]GSSG (diluted

AND ERIKSSON

with unlabeled GSSG to a specific radioactivity of about 0.04 Ci/mole), 40 mM sodium phosphate buffer (pH 7.0)-l mM EDTA, 1 mM Na,SO,, and enzyme. Isolation of GSSO,H was carried out as described above, and the total volume of the effluent (2 ml) was collected in a scintillation vial. The radioactivity was determined in a Beckman LS-100 liquid scintillation counter after addition of 15 ml scintillation fluid to the sample. The scintillation fluid consisted of 1 vol of Triton X-100 and 2 vol of toluene, containing 4 g of 2,5-diphenyloxazole and 0.1 g of 1,4-bis-2-(4.methyl5-phenyloxazolyl)-benzene per liter (10). Method C. The reaction system contained in 1 ml: 2 mM GSSG, 5 mM Na,SO,, 40 mM sodium phosphate buffer (pH 7.0), and enzyme. After incubation at 30” C the reaction was stopped by addition of 100 bl coned H,SO,. The reaction mixtures were frozen in test tubes, 1.2 ml coned H,SO, was added to the frozen samples, which were then boiled for 30 min on a water bath. The resulting thiazoline derivative was determined spectrophotometrically at 265 nm. Irrespective of the assay method, the nonenzymatic reaction was determined in parallel experiments and subtracted from the total activity found in the presence of enzyme, to evaluate the enzymatic activity.

Assays of the Reaction of GSSO,H GSH

with

Two methods were used for the study of the reaction of GSSO,H with GSH. Method D. This method is based on the determination of GSSG, which can be performed spectrophotometrically by coupling to the glutathione reductase-catalyzed reduction of GSSG with NADPH. The method has the disadvantage of being indirect, but allows registration of the reaction by a recording spectrophotometer. The reaction system contained in a final volume of 1 ml: 0.25 mM GSH, 0.25 mM GSSO,H, 0.1 mM NADPH, 0.1% human serum albumin, 0.4 unit of yeast glutathione reductase, 0.1 M sodium phosphate buffer (pH 7.0), 1 mM EDTA, and a suitable amount of enzyme. The reaction was started by addition of GSSO,H 3 min after mixing of the other components (cf. Ref. 2). The temperature of the reaction medium was maintained at 30” C. A control experiment, lacking the enzyme sample, was always carried out to determine the enzyme-independent reaction of GSSO,H with GSH. Method E. This assay method’ is based on determination of sulfite and utilizes the reaction of “Clabeled NEM with sulfite (11). The principle of the method is that the radioactivity of the NEM-sulfite derivative is determined after isolation of the latter compound. This method is direct, but does not allow continuous registration of the reaction. The reaction ZThis method has been worked out in collaboration with Mrs. M. Winell.

ENZYMATIC system contained in a final volume of 1 ml: 0.025 mM GSH, 0.28 mM GSSO,H, 0.1 M sodium phosphate buffer (pH 7.5), 0.01% human serum albumin, and the enzyme sample. Aliquots (0.2 ml) of the reaction mixture were removed after different periods of time and were mixed with 20 ~1 of 3.8 mM [“CINEM (sp act 0.36 Ci/mole). After 5 min the radioactive sample was applied to a 5 x 50-mm column of Dowex 50 W (H’) (X12, 200-400 mesh), and the sulfite adduct of NEM was then eluted with 3 ml of deionized water. The effluent was adjusted to pH 2 with 2 M HCl and remaining NEM was then extracted with 3 x 3 ml of ethyl acetate. The radioactivity was determined on a 2-ml aliquot of the water phase, mixed with 15 ml of scintillation fluid (10) (see above). RESULTS

The Reaction

of Sulfite

with GSSG

It was found that rat liver supernatants had a catalytic effect on the reaction of sulfite with GSSG (Fig. 1). The thiazoline method (8, 12), which is more specific than methods A and B for determination of GSS03H, established clearly the nature of the reaction products. The activity of 100 ~1 of crude rat liver supernatant in the assay system was lo-16 nmoles/min as determined with either of methods A and B. Boiling of the supernatant abolished the enzymatic activity, whereas gel filtration on Sephadex G-25 or dialysis for 20 hr against 5 mM sodium phosphate buffer did not. The recovery of activity after gel filtration or dialysis was normally at least 60%. To test whether any nonspecific catalysis, caused by proteins containing sulfhydry1 or disulfide groups, could contribute to the effect observed, serum albumin was tested as a possible catalyst. However, at a concentration of 10 mg/ml, which is about twice the total protein concentration used when a rat liver supernatant was assayed, no catalytic effect of serum albumin was detected. In the unfractionated supernatant, the enzymatic activity was often not proportional to the amount of enzyme added (cf. Fig. 1). This was probably due to the presence of GSH, which is present in a concentration of about 1 mM in the rat liver homogenate (cf. Table II in Ref. 1). Thus, by adding increasing amounts of enzyme,

285

SULFITOLYSIS

TIME(MIN)

FIG. 1. Sulfitolysis of GSSG catalyzed by rat liver supernatant. Reaction conditions were as described in the text (Method A). Absorbance values, corrected for a zero time control, are plotted against reaction time. (A) 50 ~1 and (0) 10 ~1 of rat liver supernatant; (0) 50 ~1 of boiled supernatant or spontaneous reaction.

which contains GSH, the starting conditions are shifted closer to the chemical equilibrium, which causes a decreased reaction rate. This explanation is supported by the observation that partially purified enzyme (see below) essentially free from GSH, demonstrated proportionality between activity and amount of enzyme. The catalytic effect was studied over a pH range of 6.0-8.0 and was found to have an optimum near pH ‘7. It should be noted that HSO,- has a pK, of 7.2, and that HSO,reacts much more slowly with GSSG than does SO,“- (13). Since GSSG does not ionize in this pH region, the acidic branch of the pH activity curve may, therefore, be related to dissociation of HSOB-, and perhaps not to ionization of a group on the enzyme. The experimental data were not extensive enough to allow proper evaluation of this suggestion. The Reaction of GSSO,H with GSH It was also found that the rat liver supernatant catalyzes the reaction of GSSO,H with GSH. This was demonstrated in the crude supernatant by assay method D, utilizing the glutathione reductase system (Fig. 2). Trace 1 of Fig. 2 shows the rate of the spontaneous reaction of GSSO,H with GSH. Trace 2 represents an experiment with crude supernatant, . in which the reaction is initiated by addition

286

MANNERVIK.

PERSSON,

GSH4

'14*+\

AND ERIKSSON

periment also demonstrates that, since glutathione reductase is not present in the assay, the reaction previously observed in the glutathione reductase-coupled system cannot be explained by possible thiol-disulfide interchange activity of the glutathione reductase preparation. Chromatographic Analysis Supernatants

FIG. 2. Reversal of the sulfitolysis of GSSG catalyzed by rat liver supernatant as demonstrated by coupling to the glutathione reductase system. The reaction was followed spectrophotometrically at 340 nm as described in the text (Method D). The traces are numbered from top to bottom, and the last addition to the cuvette is indicated at the arrow. Trace 1 represents an experiment lacking rat liver supernatant, whereas traces 2 and 3 contain 100 ~1 supernatant.

of GSSO,H. The slope corresponds to the sum of the spontaneous and the enzymatic reactions. In trace 3, the cuvette contains the same components as in trace 2, but GSH is added last instead of GSSO,H. In this case the endogenous GSH of the supernatant gives rise to a considerable reaction rate in the absence of additional GSH. When GSH is then added, however, the rate is the same as the rate represented by trace 2. The interpretation of the initial phase of trace 3 has support from the observation that partially purified enzyme, free from GSH, gave essentially no absorption change before addition of GSH, although GSSO,H was present in the cuvette. In a partially purified enzyme fraction after CM-cellulose chromatography (see Ref. 2 and below) the reaction was also measured in the absence of glutathione reductase by method E. Table I shows that in the presence of enzyme the amount of sulfite determined after 1-min incubation time is four times that in the absence of enzyme. GSH is evidently required for the time-dependent release of sulfite. The ex-

of Rat Liver

Since a catalytic effect, exerted by a partially purified enzyme catalyzing thioldisulfide interchange (2), on the reaction of GSSO,H with GSH had earlier been demonstrated (l), it was of interest to fractionate a rat liver supernatant by the technique previously used, to investigate the possibility that this enzyme was responsible for the reactions described in the present paper. A rat liver supernatant was, therefore, fractionated by gradient elution from a CM-cellulose column. Figure 3 shows that the elution profiles for the enzymatic reactions of GSH and CySSG, GSH and GSSOIH, and sulfite and GSSG each had two peaks, and that the activities coincided for the different pairs of substrates. There is evidence that the two peaks represent different forms of the same enzyme (14)) but for the present discussion it suffices to notice that the different activities have the same appearance. Furthermore, it was found that trimethylammoniTABLE

I

SULFITE FORMATION IN THE REACTION OF GSSO,H AND GSH CATALYZED BY A PARTIALLY PURIFIED ENZYMES Sulfite determined (nmoles) Incubation

Complete system Minus GSH Minus enzyme

time

I min

3 min

24.4 8.2 5.9

34.1 7.9 12.1

O1 The complete reaction system contained in a final volume of I ml: 0.025 mM GSH, 0.28 mM GSSO,H, 0.01% human serum albumin, 0.1 M sodium phosphate buffer, pH 7.5, and 0.25 mg of partially purified enzyme. The incubation was carried out at 30°C and the sulfite was determined, at the times indicated, by the [l(:]NEM method (Method E) as described in Methods.

ENZYMATIC

SULFITOLYSIS

287

umethylthiosulfate and CySSO,H could also serve as substrates for the enzyme. The coincidence of the different activities was confirmed by isoelectric focusing, and by this separation technique the glutathione reductase activity was clearly resolved from the enzyme catalyzing thioldisulfide interchange and sulfitolysis of GSSG. The recovery after CM-cellulose chromatography of the enzyme catalyzing thiol-disulfide interchange was usually

50-60%. The enzymatic activity was approximately the same with GSSO,H as with CySSG, both used at a 0.25 InM concentration. The thiosulfate esters CySSO,H and trimethylammoniumethylthiosulfate were 0 10 30 20 FRACTION NUMBER less active than GSSO,H or CySSG at 0.25 FIG. 3. Chromatographic analysis of a rat liver mM concentration in the assay system. supernatant. A rat liver supernatant was passed Both the nonenzymatic and the enzymatic through a Sephadex G-25 Fine bed after which 73 ml reactions were 2.7-fold more rapid with of the effluent was applied to a CM-cellulose column GSH and trimethylammoniumethylthio(2 x 6 cm) equilibrated with 10 mM sodium phosphate sulfate than with GSH and CySSO,H as pH 6.2, 1 mM EDTA. The elution was effected by a measured by coupling to glutathione re- linear gradient made up of 250 ml start buffer + 250 ductase. ml 50 mM sodium phosphate, pH 6.2, 0.2 M NaCl, 1 DISCUSSION

The work of Clark (15) and Lugg (16) demonstrated that low molecular-weight disulfides, such as cystine, react with sulfite to produce thiols and thiosulfate esters. This sulfitolysis is rapid and the existence of enzymatic catalysis of the reaction has consequently heretofore escaped notice. Similarly, in the case of the reverse reaction, several authors have taken the view that enzymatic catalysis is nonexistent (17-20). Only the limited observations by Siirbo (21) and by Wine11 and Mannervik (1) have earlier given evidence for an enzymatic reaction of a thiosulfate ester and a thiol. The present paper reports experiments, which for the first time show that a preparation of a biological tissue, i.e., rat liver, has the ability to catalyze both these reactions. The observations that the catalytic effect of rat liver supernatants on the sulfitolysis and its reversal was heat labile and nondialyzable are consistent with the view that the catalyst is an enzyme. The fact that the stimulation of the sulfitolysis is relatively small in the experiments described (cf. Fig. 1) should not be taken to

EDTA. The effluent was collected in lo-ml fractions. The sulfitolysis (A) was determined by Method B; the reactions of GSH and GSSO,H (A), GSH and CySSG (O), and GSH and CySSO,H (V) were determined by Method D (CySSO,H was used in 2.5-mM concentration). All activities are expressed per ml effluent. The formation of 1 pmole of product per min is used as a unit of enzymatic activity

mM

indicate that the catalysis is unimportant in vivo, because the concentration of the enzyme is considerably higher and the concentrations of the substrates will be much lower in the cell than in the reaction mixtures. For example, the GSSG concentration in rat liver is about 0.06 mM (30 pmoles/kg) (22) or probably less (23), whereas in the experiments described it was 0.4 mM. This difference would reduce the initial rate of the spontaneous reaction in vivo by a factor of 7, whereas the enzymatic reaction would be reduced to a smaller extent unless the apparent Michaelis constant for GSSG were considerably greater than 0.4 mM. The effect of the enzyme concentration will be more marked, however, because the in vivo concentration is at least 100 times the highest level in Fig. 1.

288

MANNERVIK,

PERSSON,

It is also apparent that the enzymatic sulfitolysis demonstrated may have importance for the biosynthesis of GSSO,H from GSSG and sulfite, a process as yet regarded exclusively nonenzymatic (24). Previously, it has been observed that the reversal of the sulfitolysis, i.e., the reaction of GSSO,H with GSH, was catalyzed by a partially purified enzyme, active in thioldisulfide interchange (1). The present investigation corroborates this finding, and, furthermore, shows that the two activities closely follow each other in the gradient elution of a CM-cellulose column. Thus, there is no evidence for the presence of an enzyme catalyzing the reaction of GSSO,H and GSH, which will not also use CySSG and GSH as substrates. The finding that CySSO,H or trimethylammoniumethylthiosulfate can also serve as substrates in the assay system, indicates that the enzyme catalyzing thiol-disulfide interchange is involved in the reduction of thiosulfate esters (RSSO,-) in general by the following reactions RSSO,-

+ GSH = RSSG + HSO,-

RSSG + GSH = GSSG + RSH GSSG + NADPH RSSO,-

+ H+ + 2GSH + NADP+

+ NADPH + H+ + RSH + HSO,- + NADP+

Both the first and the second of these steps are catalyzed by this enzyme as demonstrated with CySSO,H and CySSG (R = Cy), whereas the third reaction is dependent on glutathione reductase. This conclusion is at variance with earlier claims that the reaction between thiosulfate esters and glutathione is nonenzymatic (17-20). Consequently, it seems likely that the “nonenzymatic” reaction between CySSO,H and GSH, postulated in the reduction of CySSO,H in Penicillium chrysogenum (19) and Pseudomonas aeruginosa (20), is enzymatic in vivo, which implies that the enzyme catalyzing thiol-disulfide interchange is also important for the biosynthesis of cysteine via CySSO,H in these microorganisms. An enzymatic NADPH-dependent re-

AND ERIKSSON

duction of a thiosulfate ester (GSSO,H) was first described by Arrigoni and Rossi (25). This reaction was shown by Wine11 and Mannervik (1) to consist of two partial reactions, viz. (1) the formation of GSSG and sulfite from GSSO,H and GSH, and (2) reduction of GSSG mediated by glutathione reductase. The present investigation establishes the enzymatic nature of the first of these partial reactions, and the biological reduction of GSSO,H is consequently composed of two discrete enzymatic reactions catalyzed by two different enzymes. It should be pointed out that the reduction of thiosulfate esters in vivo is not necessarily linked in time to the NADPHdependent reduction of GSSG, because GSH is probably present in large excess over thiosulfate esters, a condition obviating immediate regeneration of GSH. This remark is also applicable to the reduction of disulfides such as cystine and CySSG (2). Finally, as regards the biological reduction of low molecular-weight thiosulfate esters in general, it is possible that enzymes other than glutathione reductase may catalyze the NADPH-linked reaction. Thus, the thioredoxin system (26), which is probably identical with the disulfidereducing systems earlier demonstrated by the groups of Black (27) and Wilson (28)) and with the nonspecific NADPH-dependent activity found by Tietze (29), can reduce GSSG and possibly also thiosulfate esters. However, in the analogous reduction of the mixed disulfide of coenzyme A and glutathione, which is also effected by enzymatic interchange with GSH in combination with NADPH-linked reduction of GSSG, it was concluded that the thioredoxin system could contribute only marginally to the effective reduction process (30). The same conclusion seems warranted in the case of the reduction of thiosulfate esters. Note added in proof. The reversible sulfitolysis of GSSG and the related thioldisulfide interchange described in the present paper are properly regarded as thiol-transfer reactions. We have recently proposed the name of thioltransferase for

ENZYMATIC

enzymes catalyzing this type of reactions (Askelijf, P., Axelsson, K., Eriksson, S., and Mannervik, B. (1974) Fed. Eur. Biothem. Sot. Lett. 38, 263-267). ACKNOWLEDGMENTS We thank Miss Kerstin Jacobsson for technical assistance in part of this investigation. This investigation has been supported by the Swedish Natural Science Research Council and the Swedish Cancer Society. REFERENCES 1. WINELL, M., AND MANNERVIK, B. (1969) Biochim. Biophys. Acta 184, 374-380. 2. ERIKSSON, S. A., AND MANNERVIK, B. (1970) Fed. Eur. Biochem. Sot. Lett. 7, 26-28. 3. MANNERVIK, B., Abstr. 7th Meet. Fed. Eur. Biothem. Sot. Varna 1971, p. 116. 4. ERIKSSON, B., AND RUNDFELT, M. (1968) Acta Chem. Stand. 22, 562-570. 5. ERIKSSON, B., AND EFUKSSON, S. A. (1967) Acta Chem. Stand. 21, 130441312. 6. SEGEL, I. H., AND JOHNSON, M. J. (1963) Anal.

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