From the Center of Biochemistry, Heidelberg
University, 69120 Heidelberg, Germany, the ¶ Department of
Veterans Affairs Medical Center, University of Michigan, Ann Arbor,
Michigan 48105, and the
Department of Biological Chemistry,
University of Michigan, Ann Arbor, Michigan 48105
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ABSTRACT |
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Human thioredoxin reductase is a pyridine
nucleotide-disulfide oxidoreductase closely related to glutathione
reductase but differing from the latter in having a Cys-SeCys
(selenocysteine) sequence as an additional redox center. Because
selenoproteins cannot be expressed yet in heterologous systems, we
optimized the purification of the protein from placenta with respect to final yield (1-2 mg from one placenta), specific activity (42 units/mg), and selenium content (0.94 ± 0.03 mol/mol subunit). The steady state kinetics showed that the enzyme operates by a ping-pong mechanism; the value of kcat was
3330 ± 882 min1, and the Km
values were 18 µM for NADPH and 25 µM for Escherichia coli thioredoxin. The activation energy of the
reaction was found to be 53.2 kJ/mol, which allows comparisons of the
steady state data with previous pre-steady state measurements. In its physiological, NADPH-reduced form, the enzyme is strongly inhibited by
organic gold compounds that are widely used in the treatment of
rheumatoid arthritis; for auranofin, the Ki was 4 nM when measured in the presence of 50 µM
thioredoxin. At 1000-fold higher concentrations, that is at micromolar
levels, the drugs also inhibited human glutathione reductase and the
selenoenzyme glutathione peroxidase.
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INTRODUCTION |
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Human thioredoxin reductase (NADPH + H+ + thioredoxinS2 NADP+ + thioredoxin(SH)2) is a homodimeric flavoenzyme with a
subunit size of 55.2 kDa (1-6). This enzyme and other mammalian
thioredoxin reductases have recently been shown to be selenoenzymes (2, 7-10). At present, only two other enzyme groups containing
selenocysteine are known to occur in mammals, namely glutathione
peroxidases and thyroxine deiodinases (EC 3.8.1.4) (8). Because the
presence of selenocysteine, so far, does not allow the ectopic
production of recombinant
TrxR1 (1, 8), the method for
the isolation of the enzyme from human placenta (5) was revisited and
improved with respect to speed, yield, and reproducibility.
In a previous study (7), we had investigated the reductive half-reaction of the enzyme. In brief, it was shown that the reduction of Eox, the disulfide-containing form of human TrxR, by its substrate NADPH leads to a series of transient enzyme species characterized by charge transfer complexes involving oxidized flavin, reduced flavin, and reoxidized flavin, respectively. The reactions result in a stable TrxR species containing reoxidized flavin, the active site pair Cys-57/Cys-62 as a dithiol, and an additional reduced redox active group, probably the Cys-495/SeCys-496 center. The nascent thiolate of Cys-62 forms a charge transfer complex with the flavin, which has a typical absorbance at 540 nm. Thus, human thioredoxin reductase mechanistically resembles glutathione reductase and is distinct from bacterial TrxR (7, 11, 12). Employing steady state kinetics, we have now continued investigating the catalytic mechanism of human thioredoxin reductase.
Studies with the gold compound aurothioglucose on human glutathione peroxidase (13) and human iodothyronine deiodinase type 1 (14), as well as preliminary studies on thioredoxin reductase in rat liver cytosol (15), indicate a specific inhibition of selenoenzymes by this drug. We therefore analyzed the susceptibility of isolated human thioredoxin reductase to organic gold compounds. Gold and its derivatives have been used as therapeutics in the history of mankind for ages. Most preparations and indications described were based on mystic principles and are obsolete today (16). In rheumatoid arthritis, however, a serious disease affecting 1-2% of the world's population, organic gold compounds like auranofin and aurothioglucose are still first choice therapeutics. As discussed below, the results presented here strongly suggest that gold compounds exert at least some of their pharmacologic effects by inhibiting the selenoenzyme thioredoxin reductase.
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EXPERIMENTAL PROCEDURES |
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Materials
Frozen placentas were kindly provided by Dr. J. Wacker
(Department of Obstetrics and Gynecology, Heidelberg University).
Purification of human thioredoxin reductase from placenta is delineated
below. Recombinant Escherichia coli TrxS2 with
an 280 nm of 13.7 mM
1
cm
1 (17, 18) and human glutathione reductase with an
463 nm of 11.3 mM
1
cm
1 (19) were produced and isolated as described. Human
glutathione peroxidase was purchased from Sigma.
Auranofin was obtained from ICN, and aurothioglucose, thioglucose, gold(III)chloride, and British Anti-Lewisite (BAL) (2,3-dithiopropanol) were from Sigma. Precast gels (12% polyacrylamide) and the protein dye assay were from Bio-Rad, and molecular weight standards were from Amersham Pharmacia Biotech. All reagents were of the highest available purity.
Enzyme Assays
All assays were conducted at 25 °C in a total assay volume of 1 ml.
Thioredoxin Reductase Activity--
For the purification
procedure and the inhibition studies, the DTNB reduction assay (4)
proved to be sufficiently specific. The enzyme was added to an assay
mixture of 100 mM potassium phosphate, 2 mM
EDTA, pH 7.4, and 3 mM DTNB (using a 100 mM
stock solution in Me2SO); after initiating the reaction
with the addition of NADPH (200 µM final concentration),
the increase in absorbance at 412 nm was monitored. 1 enzyme unit is
defined as the NADPH-dependent production of 2 µmol of
2-nitro-5-thiobenzoate (412 nm 13.6 mM
1 cm
1) per min.
Glutathione Reductase Activity-- Glutathione reductase activity was measured in an assay mixture consisting of 47 mM potassium phosphate, 1 mM EDTA, 200 mM KCl, pH 6.9, and 100 µM NADPH; after the addition of glutathione disulfide (1 mM final concentration), the consumption of NADPH was monitored as the decrease in absorbance at 340 nm.
Glutathione Peroxidase Activity-- Glutathione peroxidase activity was determined in a GR-coupled assay according to Beutler (20). The assay mixture (100 mM Tris-HCl, 1 mM EDTA, pH 8.0, 4 units/ml glutathione reductase, 2 mM reduced glutathione, 100 µM NADPH, and glutathione peroxidase) was equilibrated for 10 min; then the substrate t-butylhydroperoxide (1 mM final concentration) was added and the consumption of NADPH was monitored. We increased the activity of glutathione reductase in the assay from 1 unit/ml (20) to 4 units/ml to assure that this ancillary enzyme was not rate-limiting in the presence of organic gold compounds.
Protein Assay-- Protein was determined using the Bio-Rad dye assay with bovine serum albumin as a standard.
Thioredoxin Reductase Purification
Because of the potential risk of infection, laboratory biosafety regulations (3, 21) were strictly obeyed in the first steps including acetone precipitation. Unless otherwise stated, all procedures were carried out at 4 °C. The TE buffer used throughout the preparation consisted of 50 mM Tris-HCl, 1 mM EDTA, pH 7.6.
Chloroform-1-butanol Extraction--
(22)A frozen placenta of
approximately 500 g was cut with a stainless steel saw into slices
(about 1 × 3 × 10 cm). The slices were cleaned mechanically
from debris with a cover slide, weighed out, and transferred to plastic
bags. Per 1 g of placenta, 0.6 ml of extraction solution (10 µM FAD, 40 µM phenylmethylsulfonyl fluoride
in TE buffer) was added, and the tissue was thawed by placing the bags
into a 40 °C water bath. Subsequently, the content of the bags was
homogenized in 250-g portions in a Waring blender. Each portion was
stabilized with 20 µl of 100 mM phenylmethylsulfonyl fluoride. Immediately before treatment with chloroform/1-butanol, the
homogenate was titrated to pH 8.3 using 5 M
NH4OH. The chloroform/1-butanol mixture (1:2.5, v/v;
20 °C; 120 µl/gram placenta) was added under vigorous stirring.
The brownish suspension was rehomogenized in the Waring blender, left
for 1 h, and then centrifuged for 90 min at 8000 × g. The supernatant was set aside while the precipitate was
taken up in extraction solution (0.4 ml/gram placenta), homogenized, and centrifuged as above. The supernatants were combined, filtered through glass wool (Riedel de Häen) and adjusted to pH 8.3 using 5 M NH4OH. This solution was the
chloroform-butanol extract (Table I).
Acetone Precipitation-- Per 1 ml of chloroform-butanol extract, 0.85 ml of acetone was slowly added under stirring. The solution was left for 1 h and then centrifuged for 15 min at 3500 × g. The pellet was taken up in a small volume of TE buffer having a final volume of approximately 100 ml. This sample was dialyzed exhaustively against 2-fold diluted TE buffer and centrifuged (30 min, 25000 × g). The supernatant was set aside while the pellet was resuspended in TE buffer, mixed carefully, and centrifuged as described above. The combined supernatants were filtered through glass wool and adjusted to pH 8.3 using 5 M NH4OH. The resulting solution was referred to as the acetone-treated fraction.
DEAE-52 Cellulose Chromatography-- The acetone-treated fraction was applied to a (3.2 × 50 cm) DEAE-52 cellulose column (Whatman), which had been equilibrated with TE buffer before and was operated at room temperature. After washing the column with 1000 ml of TE buffer followed by 500 ml of 50 mM NaCl in TE buffer, thioredoxin reductase activity was eluted with 90 mM NaCl in TE buffer. In this step, the enzyme comigrated with a deep red protein. The pool of active fractions was concentrated and washed with TE buffer in a Centriprep 30 (Amicon). The resulting solution was diluted 2-fold with TE buffer, and the pH was adjusted to 7.6 using 100 mM HCl. This fraction was called the DEAE-cellulose eluate.
2',5'-ADP-Sepharose 4B Affinity Chromatography-- The above fraction was applied to a 30-ml (1.5 × 17 cm) 2',5'-ADP-Sepharose 4B column (Amersham Pharmacia Biotech) in a jacketed chromatography tube. The tube was cooled to 6 ± 1 °C, the exact temperature being crucial for the purification success. The column was consecutively washed with 60 ml of TE buffer, 30 ml of 100 mM KCl in TE buffer, 20 ml of 200 mM KCl in TE, 30 ml of 100 mM KCl in TE, 60 ml of 2-fold diluted TE, 60 ml of 500 µM NADH in TE, 60 ml of TE, 60 ml of 100 µM NADP+ in TE, and 30 ml of 300 µM NADP+ in TE. Finally, TrxR activity was eluted with 750 µM NADP+ in TE buffer, concentrated, and washed with the buffer in a Centriprep 30. This solution, the 2',5'-ADP-Sepharose eluate, contained (on the basis of absorption spectra (7), specific activity, and SDS-polyacrylamide gel electrophoresis analysis) homogeneous (more than 95% pure) thioredoxin reductase.
Sephadex G-200 Gel Filtration-- To remove trace impurities, the above fraction may be applied to a Sephadex G-200 column (Amersham Pharmacia Biotech, 1 × 100 cm) equilibrated with TE buffer. Spectroscopically pure fractions were pooled, concentrated, and referred to as the Sephadex G-200 eluate.
Inhibitor Studies
10 mM stock solutions of aurothioglucose and thioglucose in assay buffer and of auranofin in Me2SO were prepared immediately prior to use and stored in dark bottles. Dilutions were made in assay buffer. One min after adding the inhibitor to the assay mixture of a given enzyme, the reaction was started with the appropriate substrate. Thioglucose served as a control and had no inhibitory effect on the enzymes within the tested inhibitor concentration range.
For inhibition studies on human TrxR, the DTNB reduction assay was used. To verify the effects of an organic gold compound on TrxR under more physiological conditions, we determined the Ki for auranofin with TrxS2 as a substrate. Using two different TrxS2 concentrations (50 and 75 µM together with 100 µM NADPH and 1.7 nM TrxR), the inhibitory effects of 5-50 nM auranofin in the assay were observed over 20 min, and initial rates were determined.
To determine the influence of NADPH on TrxR and glutathione reductase inhibition by the gold compounds, 50-µl samples containing approximately 600 nM enzyme subunits each were preincubated with 1 µM inhibitor in the presence and absence of 200 µM NADPH for 20 min at room temperature. Aliquots were taken, diluted, and assayed for residual activity. Control experiments showed that free inhibitor present in diluted aliquots had no effect when added to assays of uninhibited enzyme. To test the reversibility of TrxR inhibition by a gold chelating agent, inhibited samples were exposed to 1 mM BAL for 5 and 20 min before assaying activity. In a complementary protection experiment, 50-µl samples of TrxR (700 nM subunits) containing 200 µM NADPH and 1 mM BAL were prepared. Then inhibitor was added (1 µM), and after 20 min the residual activity was determined.
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RESULTS |
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Enzyme Purification-- Because it is not yet possible to produce recombinant mammalian selenoenzymes in heterologous systems, we have optimized the purification of native human thioredoxin reductase. For this purpose, placenta proved to be the organ of choice (5). The first purification steps involve organic solvents (Table I). Apart from their antiseptic effect, these solvents denature the bulk of NADP(H)-dependent enzymes (22), which greatly enhances the efficiency of affinity chromatography used in a later purification step. In comparison with the original report of Oblong et al. (5), we were able to improve the isolation procedure with respect to speed, final yield (1-2 mg of TrxR instead of 0.3 mg from one placenta) and specific activity (Table I). Using atomic absorption spectroscopy, the selenium content of the isolated enzyme was found to be 0.94 ± 0.03 mol/mol of subunit of 55.2 kDa. Approximatly 520 pmol of TrxR subunit exhibits 1 unit of enzymatic activity in the DTNB reduction assay.
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Kinetic Studies--
The long-standing problem of preparing the
substrate thioredoxin in sufficient amounts has led in the past to the
use of DTNB as the disulfide substrate or to the use of coupled
disulfide systems in TrxR assays (23). Comparisons of these alternative systems with thioredoxin-based assays have been conducted whenever it
was crucial (23, 24). Luthman and Holmgren found that rat liver, calf
liver, and E. coli thioredoxin all gave a
kcat of approximately 3000 min1
with rat liver thioredoxin reductase; using DTNB as the acceptor substrate, kcat was 4000 min
1
(24). In the present study on human TrxR, we have used E. coli thioredoxin (TrxS2) and found, as detailed below,
a turnover number of 3300 min
1.
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Inhibitor Studies-- The results of the studies with aurothioglucose and auranofin in the assay mixture are shown in Fig. 3. Gold-free thioglucose did not inhibit the enzymes in the concentration range used for the gold compounds. Glutathione reductase and glutathione peroxidase were by at least three orders of magnitude less susceptible to the organic gold compounds than thioredoxin reductase. IC50 values for the inhibition of all three enzymes by the different inhibitors are given in Table II. It should be noted that glutathione reductase was almost unaffected by aurothioglucose, whereas auranofin had inhibitory effects although only in the upper micromolar range. For glutathione peroxidase, the situation was reversed; that is, auranofin but not aurothioglucose was found to be an inhibitor. Qualitatively speaking, our data agree well with the effects of aurothioglucose on the three enzymes in unfractionated rat cytosol. In this study, TrxR was inhibited 50% by 100-fold less gold thioglucose than needed for 50% inhibition of glutathione peroxidase, whereas GR activity was not affected by submillimolar concentrations of the gold compound (15).
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
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DISCUSSION |
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Isolation of Authentic hTrxR-- Difficulty in preparing recombinant hTrxR is a major problem in studying the human thioredoxin system. The published purification protocols for placenta hTrxR result in comparatively low yields and require many time consuming steps (5). The efficient purification procedure presented here provides sufficient amounts of thioredoxin reductase for structural studies. We recently succeeded in crystallizing the isolated enzyme by using PEG 8000 in Tris buffer of pH 7.4 as a precipitant.
Kinetic Studies--
The steady state kinetic data are consistent
with a bi-bi-ping-pong mechanism, a result which further underlines the
similarities between human thioredoxin reductase and glutathione
reductase (7, 12, 26). Using the Arrhenius diagram (Fig. 2), the apparent turnover number of hTrxR at 4 °C is 650 ± 17 min1 or 10.8 ± 2.9 s
1. This allows
the comparison of the steady state kinetic data with the
presteady-state rates determined at 4 °C for the reduction of hTrxR
by its substrate NADPH (7); the low temperature was necessary because
the first two phases of this reaction are very fast. A rapid absorbance
increase at 540 nm (110 s
1), for instance, reflected the
reduction of the active site disulfide Cys-57/Cys-62 as indicated by a
charge transfer complex between the nascent thiolate 62 with FAD. The
slowest reaction phase was observed as an absorbance
decrease at 540 nm, signaling reformation of the active site
disulfide; it occurred at a rate of approximately 5 s
1,
which is comparable with the turnover number of TrxR at 4 °C (Fig.
2). On the basis of these data, it is tempting to speculate that
kcat of human TrxR is limited at least in part
by redox interchange between the active site Cys-57/Cys-62 pair and the
Cys-495/SeCys-496 redox center. The notion that the active site dithiol
passes the reducing equivalents on to another redox center is supported
by the observation that at least two equivalents of reducing agent (carrying four electrons) are needed for the complete reduction of the
active site disulfide (7).
Gold Compounds as Inhibitors-- Aurothioglucose and auranofin were found to be potent inhibitors of human thioredoxin reductase (Table II, Equations 2 and 3). These organic gold compounds are widely used in the treatment of rheumatoid arthritis. The disease is considered to be an autoimmune condition initiated by various agents, the Epstein-Barr virus being the prime candidate (31). Lymphocytes infected with EBV or other viruses have been shown to secrete thioredoxin (32) which, together with our data, suggests the possibility that the thioredoxin redox system plays a prominent role in autoimmune processes. This notion is supported by the finding that in Sjögren's syndrome, another autoimmune disease with joint involvement, secreted thioredoxin levels correlate very well with the expression of EBV material (34). With respect to the inhibition studies on TrxR, it should be emphasized that the activity of thioredoxin as a cytokine depends on its reduced dithiol state (32, 33).
As shown in Fig. 4, NADPH-reduced human thioredoxin reductase is highly sensitive to gold compounds, whereas the oxidized form of the enzyme, Eox, is not affected. Since the Km value (= Kdiss value) for NADPH under quasi in situ conditions is 3-5 times lower than the cytosolic NADPH concentration (7), the reduced gold-sensitive forms of TrxR are likely to be predominant in situ. Not only chemical but possibly also steric reasons may account for the different sensitivities of oxidized and reduced TrxR. For the three human iodothyronine deiodinases, all of them being selenoenzymes, it has been shown that the type 1 enzyme is strongly inhibited by aurothioglucose (Ki, app ~5 nM), whereas the type 2 and type 3 enzymes are 1000-fold less sensitive (38). Several lines of reasoning indicate, that it is indeed the gold content of the compounds which leads to thioredoxin reductase inhibition. First, the thioglucose moiety of aurothioglucose (and auranofin) is not an inhibitor in the concentration range used in our study. Second, the gold-chelating agent BAL is able both to prevent and to reverse the inhibition of TrxR caused by three different compounds that have only the gold moiety in common. Furthermore, selenols exhibit a higher tendency to bind heavy metal ions than thiols do (16). It is therefore tempting to speculate that the C-terminal redox-active Cys-495/SeCys-496 center of thioredoxin reductase is the target of the inhibitors (39, 40). This view is supported by the finding that the structurally and mechanistically closely related but selenium-free enzyme glutathione reductase (1, 7, 9) is far less sensitive to the inhibition by auranofin and aurothioglucose (Fig. 3 and Ref. 15). Inorganic AuCl3 (which is not in therapeutic use because of its high toxicity) caused a BAL-resistant inhibition of NADPH-reduced GR. This indicates that, at least for GR, the mode of inhibition by inorganic Au(III)compounds is different from drugs like auranofin and aurothioglucose that contain an Au(I)-moiety. Virtually complete TrxR inhibition in vitro can be achieved with concentrations far below the clinically used plasma levels (e.g. 20 µM for auranofin). Under in vivo conditions, the drug is likely to be bound unspecifically to compounds such as glutathione and other thiols which decreases the actual concentration of free inhibitor. This interpretation is supported by the observation that a 10-fold higher aurothioglucose concentration is needed for 50% TrxR inhibition in rat liver cytosol (15) when compared with our data on the isolated human enzyme. Auranofin has also been successfully tested as an antineoplastic agent which efficiently inhibits DNA synthesis (Ref. 35 and references therein). Because intracellular thioredoxin is a reducing substrate of ribonucleotide reductase, a key enzyme in DNA synthesis, our results can offer a molecular explanation also for this effect. Other studies dealing with pharmacological effects of carmustine (3, 7, 37), with the abundancy of TrxR activity in tumor cells (2), with NK lysin as a substrate of TrxR (36), or with reduced thioredoxin as a proliferation-promoting cytokine (32, 33) indicate as well that inhibition of thioredoxin reductase may be a rational approach to the treatment of certain malignancies. ![]() |
ACKNOWLEDGEMENTS |
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We are grateful to Irene König and Donna Veine for assistance with the protein purification.
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FOOTNOTES |
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* The study was funded by the Deutsche Forschungsgemeinschaft (Schi 102/7-5 to R. H. S.), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Research Focus Tropical Medicine, 01 KA 9301 to K. B.), and the Department of Veterans Affairs and the National Institute of General Medical Sciences Grant GM21444 (to C. H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Studienstiftung des Deutschen Volkes.
** To whom correspondence should be addressed: Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. Tel.: 49 6221 54 4175; Fax: 49 6221 54 5586; E-mail: schirmer.{at}urz.uni-heidelberg.de.
The abbreviations used are: TrxR, thioredoxin reductase; BAL, British Anti-Lewisite (2,3-dithiopropanol); DTNB, 5,5'-dithiobis-(2-nitrobenzoate); EBV, Epstein-Barr virus; Eox, oxidized thioredoxin reductase containing an active site disulfideGR, glutathione reductaseSeCys, selenocysteineTE, 50 mM Tris-HCl, 1 mM EDTA adjusted to pH 7.6 at 25 °CTrxS2, E. coli thioredoxin in oxidized form.
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REFERENCES |
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