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Originally published In Press as doi:10.1074/jbc.M112115200 on February 26, 2002

J. Biol. Chem., Vol. 277, Issue 20, 17548-17555, May 17, 2002
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Catalytic Properties, Thiol pK Value, and Redox Potential of Trypanosoma brucei Tryparedoxin*

Nina Reckenfelderbäumer and R. Luise Krauth-SiegelDagger

From the Biochemie-Zentrum Heidelberg, Universität Heidelberg, 69120 Heidelberg, Germany

Received for publication, December 19, 2001, and in revised form, February 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The dithiol protein tryparedoxin is a component of the unique trypanothione/trypanothione reductase metabolism of trypanosomatids and is involved in the parasite synthesis of deoxyribonucleotides and the detoxication of hydroperoxides. Tryparedoxin is a highly abundant protein in all life stages of Trypanosoma brucei, the causative agent of African sleeping sickness. As shown here, its functional properties are intermediate between those of classical thioredoxins and glutaredoxins. The redox potential of T. brucei tryparedoxin of -249 mV was determined by protein-protein redox equilibration with Escherichia coli thioredoxin. The trypanothione/tryparedoxin couple is probably the most significant factor determining the cytosolic redox potential of the parasites. The pK value of Cys40, the first thiol in the WCPPC motif, is 7.2 as derived from the thiolate absorption at 240 nm and the rate of carboxymethylation. Alteration of the active site into that of thioredoxin (CGPC) did not affect the pK value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the pK dropped to <= 4.0. The fact that the pK value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tryparedoxins are 16-kDa dithiol proteins that belong to the large family of thioredoxin-like thiol-disulfide oxidoreductases, all of which share a CXXC active site motif by low overall sequence similarities. The redox active motif reads CPPC in tryparedoxins (1, 2), CGPC in thioredoxins (3, 4), CPYC in glutaredoxins (5, 6), CGHC in eukaryotic protein-disulfide isomerases, and CPHC in the bacterial periplasmatic protein thiol:disulfide oxidoreductase DsbA (7).

So far tryparedoxins have been found only in parasitic protozoa of the family Trypanosomatidae, to which belong the causative agents of tropical diseases such as African sleeping sickness (Trypanosoma brucei gambiense and T. brucei rhodesiense), Nagana cattle disease (T. brucei and Trypanosoma congolense), Chagas' disease (Trypanosoma cruzi), and the three manifestations of leishmaniasis (Leishmania donovani, Leishmania major, and Leishmania mexicana). All of these parasitic protozoa have in common that they lack the ubiquitous glutathione/glutathione reductase system but have a trypanothione [bis(glutathionyl)spermidine]/trypanothione reductase system instead (8-10). In addition, trypanosomes do not have catalase and classical selenocysteine-glutathione peroxidase. Their known sensitivity toward oxidative stress renders the enzymes of the trypanothione metabolism attractive targets for a rational drug development (9).

Initially tryparedoxin was isolated from the insect parasite Crithidia fasciculata (1), but the protein occurs also in the pathogenic trypanosomatids T. brucei (2), T. cruzi (11), and L. major (12). Its first elucidated role was as a component of a cascade composed of trypanothione, trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase that catalyzes the detoxication of organic hydroperoxides in the parasites (1, 13). We have cloned and overexpressed the gene encoding tryparedoxin from T. brucei (2). The recombinant protein catalyzes the trypanothione-dependent reduction of ribonucleotide reductase during the parasite synthesis of DNA precursors (14). In the latter reaction, tryparedoxin thus can replace the well known thioredoxin and/or glutaredoxin systems in other organisms (5). T. brucei also possesses a classical thioredoxin, but its concentration is unusually low (15). A thioredoxin reductase has not been detected so far in any Trypanosomatid organism. Preliminary functional studies showed that tryparedoxin has properties intermediate between those of classical thioredoxins and glutaredoxins. Like thioredoxin, tryparedoxin shows a negligible activity in the GSH:hydroxyethyl disulfide transhydrogenation, a reaction characteristic for glutaredoxins (2). With glutaredoxins tryparedoxin has in common that the physiological reductant is a nonprotein thiol.

The three-dimensional structure of C. fasciculata tryparedoxin revealed a folding very similar to that of thioredoxins into a five-stranded beta -sheet surrounded by four alpha -helices (16, 17). In particular the active site motif Cys40-Pro41-Pro42-Cys43 is in a position homologous to that of the corresponding motif in thioredoxin. The side chain of Cys40 points out of the protein being accessible from the solvent; in contrast, Cys43 is much more buried. Cys40 is the catalytic residue interacting alternately with trypanothione and the protein substrate such as tryparedoxin peroxidase or ribonucleotide reductase.

In all thioredoxin-like proteins the reactivity is determined by the pK value of the first cysteine residue in the CXXC motif as well as by the redox potential of the protein. The first thiol is the nucleophile that reacts with the respective substrate. Here we report on the catalytic activities, the determination of the pK value of Cys40, and the redox potential of T. brucei tryparedoxin. Two active site mutants, mimicking the motif of classical thioredoxins and glutaredoxins, respectively, were constructed and compared with the authentic protein.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Trypanothione disulfide was purchased from Bachem. The reduced form, trypanothione, T(SH)21 was prepared by NaBH4 reduction as described (14). Dehydroascorbate was from Serva. 20 mM stock solutions were prepared in H2O and stored at -80 °C. Escherichia coli glutaredoxin was obtained from IMCO, and E. coli thioredoxin was from Calbiochem. A sample of human thioredoxin reductase was a kind gift of Drs. Katja Becker (Universität Giessen) and R. Heiner Schirmer (Biochemie-Zentrum Heidelberg). T. brucei tryparedoxin (2), T. cruzi trypanothione reductase (18), and T. brucei thioredoxin (15) were prepared as described. Partially purified malate dehydrogenase from spinach was a kind gift of Dr. Hartmut Follmann (Universität Kassel) and was stored at -20 °C. Polyclonal rabbit antibodies against recombinant T. brucei tryparedoxin were produced by Eurogentec.

Reduction of Dithiol Proteins by NaBH4-- In a total volume of 400 µl, 200 µM T. brucei tryparedoxin or E. coli thioredoxin was incubated with 20 mM NaBH4 in 100 mM potassium phosphate, pH 7.0, for 5 min at room temperature. HCl was added to destroy excess NaBH4, and the protein solutions were desalted on a PD10 column (Amersham Biosciences). Complete reduction of the proteins was confirmed by HPLC analysis as described below and by thiol determination with Ellman's reagent (19).

Alkylation of Tryparedoxin-- Carboxamidomethylation of tryparedoxin was carried out as described (14). Carboxymethylated tryparedoxin was prepared by incubating 50 µM tryparedoxin with 1 mM DTE in 50 mM Hepes, pH 7.6. After adding 6 mM iodoacetic acid, the reaction was allowed to proceed for 1 h at room temperature in the dark. The reaction was stopped by excess DTE. To prepare tryparedoxin alkylated at both active site cysteines, the reaction was carried out in the presence of 6 M guanidinium chloride.

Glutathione-dependent Dehydroascorbate Reductase Assays-- The reaction mixtures contained in a total volume of 80 µl of 100 mM potassium phosphate, 1 mM EDTA, pH 6.5, 100 µM dehydroascorbate, 1 mM GSH, and 0.1-5.6 µM T. brucei tryparedoxin, E. coli glutaredoxin, and thioredoxin, respectively. Formation of ascorbate was followed at 25 °C (epsilon 265 = 14,000 M-1 cm-1) in a Beckman DU-65 spectrophotometer (20). The protein-mediated activity was corrected for the spontaneous reaction rate.

pH Dependence of the Tryparedoxin Catalyzed Reduction of Dehydroascorbate by GSH-- Reduction of 100 µM dehydroascorbate by 1 mM GSH was followed in the presence and absence of 2 µM tryparedoxin in a total volume of 80 µl of 0.1 M MOPS/NaOH (pH 6.0-7.5) and Tris/HCl (pH 8.0-8.5) containing 0.2 M KCl, 1 mM EDTA at 25 °C.

Reduction of Dehydroascorbate by Trypanothione-- In a total volume of 80 µl, the reaction mixtures contained 50 and 100 µM dehydroascorbate, respectively, in 100 mM potassium phosphate, 1 mM EDTA, pH 6.5. The reaction was started by adding 5-50 µM T(SH)2, and formation of ascorbate was followed as described above. A second assay system contained 50 or 100 µM dehydroascorbate, 10 or 50 µM T(SH)2, 100 µM NADPH, 10 milliunits trypanothione reductase, and 1.1-3.3 µM T. brucei tryparedoxin.

Malate Dehydrogenase Assay-- Partially purified malate dehydrogenase from spinach was freed of plant thioredoxins immediately before use. At 4 °C 2 ml of protein solution was applied onto a Sephadex G-50 column (1.2 cm × 89 cm) in 200 mM potassium phosphate, pH 7.0, and eluted at a flow rate of 0.3 ml/min. 4-ml fractions were collected, and the protein concentrations of the first 30 fractions were determined at 280 nm. Fractions containing more than 1 mg/ml protein were combined giving 32 ml of partially purified malate dehydrogenase with a protein concentration of 1.8 mg/ml. In a total volume of 800 µl of 100 mM Tris/HCl, pH 7.9, 0.1-20 µM E. coli thioredoxin and glutaredoxin as well as T. brucei tryparedoxin and thioredoxin, respectively, were incubated with 10-80 µl of malate dehydrogenase solution and 5 mM DTE for 30 min at 25 °C. The reaction was started by adding 2.5 mM oxaloacetate and 0.2 mM NADPH, and the absorption decrease at 340 nm was followed in a Hitachi 150-20 spectrophotometer (21).

Construction of Active Site Mutants-- The WCPPC active site motif of tryparedoxin was altered into the sequence of classical thioredoxins (WCGPC) and glutaredoxins (WCPYC). For each mutant forward and reverse primers were derived covering the active site (thioredoxin-mutant: reverse, 5'-CCCCGGCATGGGCCGCACCAGGAGGC-3', and forward, 5'-GGTGCGGCCCATGCCGGGGTTTTACACCGG-3'; glutaredoxin-mutant: reverse, 5'-CCCCGGCAATAGGGGCACCAGGAGGC-3', and forward, 5'-CCCCTATTGCCGGGGTTTTACACCGGTCC-3'). The codons inducing the mutation are underlined. The primers overlapped by 15-19 base pairs. Three polymerase chain reactions were carried out with Pfu polymerase (at 95 °C for 2 min; 30 times at 95 °C for 1 min, at 55 °C for 1 min, and at 72 °C for 1 min; and at 72 °C for 5 min). In the first amplification the respective active site reverse primer and an upstream pQE-60 vector primer (5'-GAGCGGATAACAATTTCACACAGAATTC-3') were used. The second PCR was carried out with the active site forward primer and a primer corresponding to the 3'untranslated region directly following the stop codon (5'-AGATCTTCACAGACAGCATGGCATCTC-3', with a BglII cleavage site underlined). The pQE-60 plasmid with the tryparedoxin coding region served as template. In the third PCR the two purified fragments were used together as template, and the complete tpx gene with the respective mutation was amplified with the 3'-end primer and the pQE-60 vector primer. The PCR fragments were purified, cloned into a pQE-60 vector, and completely sequenced in both directions. The mutant genes were expressed in the thioredoxin-deficient E. coli strain A 179, and the proteins were purified as described for authentic T. brucei tryparedoxin (2). CD spectra of wild type and mutant proteins were recorded on a Jasco J-710 spectropolarimeter at the EMBL (Heidelberg, Germany) in collaboration with Drs. Manuela López de la Paz and Luis Serrano.

Reduction of Tryparedoxin by Human Thioredoxin Reductase-- The reaction mixture contained in a total volume of 80 µl of 100 mM potassium phosphate, 2 mM EDTA, pH 7.4, 100 µM NADPH, human thioredoxin reductase, and varying concentrations of T. brucei tryparedoxin and the mutants, respectively (5-30 µM). The absorption decrease at 340 nm was followed at 25 °C in a Beckman DU-65 spectrophotometer. The Km values of human thioredoxin reductase for the different T. brucei tryparedoxin species were derived from Lineweaver-Burk plots. Relative maximum activities were determined because the available sample of human thioredoxin reductase was not homogeneous.

Measurement of the Thiolate Anion by UV Absorption-- The pH-dependent ionization of the cysteine 40 thiol was monitored by the absorbance of the thiolate anion at 240 nm (7). Spectra of 10 µM reduced, oxidized, and carboxamidomethylated tryparedoxin were recorded between 200 and 400 nm at 25 °C in a Beckman DU-7400 spectrophotometer. All of the measurements were carried out in a stoppered quartz cuvette that contained in a total volume of 1.3 ml of 10 µM tryparedoxin, 1 mM each citrate, borate, and phosphate, 0.2 M KCl, pH 5.2, purged with argon. The pH value was adjusted with KOH and then raised in steps between pH 5.2 and 10 by adding defined volumes of 0.2 M KOH. The spectra were recorded against air and corrected for the dilution caused by the pH adjustment, and the spectra of the buffer solutions treated in the same way were subtracted. In the case of the glutaredoxin mutant, the solution was titrated between 5.2 and 7.05 with KOH and between 4.8 and 2.5 by adding HCl.

Rate of Carboxymethylation of Cysteine 40-- 50 µM reduced tryparedoxin was incubated at 25 °C with 50 µM iodoacetic acid in 100 µl of 10 mM Tris, 10 mM potassium acetate, 10 mM MOPS, 10 mM MES, 0.2 M KCl at pH values between 5.9 and 9.5 (7, 22). After different times, the reaction was stopped by mixing 10 µl of the sample with 10 µl of 200 mM DTE. Reduced and carboxymethylated tryparedoxin were separated by HPLC on a C8-VYDAAC column (208TP5215) at 25 °C. The proteins were detected at 214 nm and eluted isocratically with 41% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.2 ml/min.

Determination of the Redox Potential-- The redox potential of T. brucei tryparedoxin was determined by direct protein-protein redox equilibration (23). Different concentrations of reduced and oxidized tryparedoxin and E. coli thioredoxin in 100 µl of 100 mM potassium phosphate, pH 7.0, were allowed to equilibrate for 4 h at 25 °C in a closed 1.5-ml reaction tube after purging the solution with argon. 5-µl samples were directly analyzed by HPLC on a C8-VYDAAC column (208TP5215). The proteins were eluted over 50 min by a gradient from 35.5 to 70% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.2 ml/min and 25 °C.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reduction of Dehydroascorbate in T. brucei-- In vitro activity of glutaredoxins (thioltransferases) is the ability to catalyze the reduction of dehydroascorbate by glutathione (24). Thioredoxins do not catalyze the reaction whereby a recent report shows that the mammalian thioredoxin reductase/thioredoxin system can reduce dehydroascorbate (25). Because T. brucei tryparedoxin has properties intermediate between those of classical glutaredoxins and thioredoxins, we tested whether the parasite protein is able to accelerate the spontaneous reduction of dehydroascorbate by glutathione. The reaction was followed by measuring the formation of ascorbate. Because of the rapid nonenzymatic reduction of dehydroascorbate by GSH at higher pH values, the assays were performed at pH 6.5 (20). The reaction mixtures contained 100 µM dehydroascorbate, 1 mM glutathione, and varying concentrations of T. brucei tryparedoxin, E. coli glutaredoxin, and E. coli thioredoxin, respectively. 2 µM tryparedoxin catalyzed the formation of ascorbate with a rate of 0.09 µM min-1 (Fig. 1), which is 1 order of magnitude slower than the reaction mediated by 2 µM E. coli glutaredoxin (0.94 µM min-1). As expected, E. coli thioredoxin showed only marginal activity. Reduction of dehydroascorbate by glutathione is strongly dependent on the pH value. Between pH 6.5 and 7.5, the rate of the spontaneous reaction increases by about 1 order of magnitude. In contrast, the slight increase in the reaction rate in the presence of tryparedoxin remained constant. As shown previously, trypanothione reduces dehydroascorbate at pH 6.5 with a second order rate constant of 1300 M-1 min-1 (20). The reaction is 3 orders of magnitude faster than reduction by glutathione. A further acceleration of the reaction rate by tryparedoxin was not observed, in accordance with ascorbate being kept reduced by the spontaneous reaction with trypanothione.


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  		Fig. 1.   Reduction of dehydroascorbate by glutathione. The reaction mixture contained 100 µM dehydroascorbate and 1 mM GSH in 100 mM potassium phosphate, 1 mM EDTA, pH 6.5, and varying concentrations of T. brucei tryparedoxin (), E. coli glutaredoxin (open circle ), and E. coli thioredoxin (black-down-triangle ). The rates were corrected for the spontaneous reduction of dehydroascorbate by glutathione under these conditions (0.11 µM min-1). The values depicted are the means of two independent measurements that varied by less than 5%.

Activation of NADP-Malate Dehydrogenase-- NADP-dependent malate dehydrogenases from plants are activated by a thiol/disulfide interchange with reduced thioredoxins. Reduction of oxaloacetate by partially purified malate dehydrogenase from spinach chloroplasts was followed in the presence of T. brucei tryparedoxin and T. brucei thioredoxin. E. coli thioredoxin, known to activate NADP-malate dehydrogenase and E. coli glutaredoxin, not catalyzing the reaction (Fig. 2), served as control. The preincubation mixture contained 5 mM DTE, which does not activate malate dehydrogenase directly but only serves to generate the reduced dithiol proteins in the assay (21). In the presence of T. brucei thioredoxin, spinach malate dehydrogenase showed a sigmoidal saturation curve, whereas E. coli thioredoxin yielded a Michaelis-Menten-type kinetic. The presence of both proteins resulted in identical maximum activities. T. brucei tryparedoxin activated the enzyme with a sigmoidal dependence on the protein concentration, but maximum activity was not achieved. The tryparedoxin concentration required for half-maximum activity was about an order of magnitude higher when compared with the thioredoxins. As expected, E. coli glutaredoxin failed to activate malate dehydrogenase. The finding that tryparedoxin and the two thioredoxins exert different degrees and types of activation is consistent with the reaction not being the simple reduction of malate dehydrogenase. Obviously specific complex formation between the dithiol protein and the enzyme is involved in the activation mechanism (21). A thorough study on spinach and soybean NADP-malate dehydrogenase showed that the activation and activity of the enzymes strongly depend on the individual thioredoxin, with the kinetics varying from Michaelis-Menten-type to different sigmoidal kinetics (21).


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  		Fig. 2.   Activation of NADP-dependent malate dehydrogenase by dithiol proteins. Partially purified malate dehydrogenase was preincubated in 100 mM Tris/HCl, pH 7.9, for 30 min at 25 °C with 5 mM DTE and different concentrations of E. coli thioredoxin () and T. brucei thioredoxin (open circle ) (A) and T. brucei tryparedoxin () and E. coli glutaredoxin (open circle ) (B). The reaction was started by adding 2.5 mM oxaloacetate and 0.2 mM NADPH. The values are the means of four independent measurements that differed by less than 10%. The data points were fitted to a hyperbola (E. coli thioredoxin) and sigmoidal curves (T. brucei thioredoxin and tryparedoxin) using the regression program of Sigma plot 4.0.

Generation of Active Site Mutants-- Two active site mutants of T. brucei tryparedoxin were constructed corresponding to the motif of classical thioredoxins (CGPC) and glutaredoxins (CPYC), respectively. The mutant genes were overexpressed in thioredoxin-deficient E. coli as described for wild type tryparedoxin (2). 20 mg of pure protein was obtained per liter bacterial cell culture, the yield being comparable with wild type tryparedoxin. Correct folding of the mutant proteins was examined by CD spectroscopy. The shape of the spectra was very similar to that of authentic tryparedoxin (not shown). Calculating the ratios of the mean residue ellipticity values at 208 and 222 nm as well as at 217 and 206 nm yielded identical values, indicating the same content of alpha -helices and beta -sheets in the three protein species.

Reduction by Human Thioredoxin Reductase-- Mammalian thioredoxin reductases show a broad substrate specificity accepting thioredoxins from different species. T. brucei tryparedoxin is reduced by human thioredoxin reductase with a Km value of 43.5 µM (Table I), which is comparable with the Km values of 20 and 6 µM for E. coli and T. brucei thioredoxin, respectively (15). Changing the active site motif into that of thioredoxin lowered the Km value to 17.4 µM but also decreased the reaction rate. The mutant with the WCPYC sequence of glutaredoxin was also a substrate for human thioredoxin reductase with a very high Km value of 200 µM but with a concomitantly increased activity. The simultaneous increase/decrease of the Km value and the activity resulted in catalytic efficiencies for the two mutants only 30-40% lower than that of wild type tryparedoxin.

                              
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Table I
Kinetic parameters for the reduction of different T. brucei tryparedoxin species by human thioredoxin reductase
The assays were carried out as described under "Experimental Procedures," and the kinetic data were derived from Lineweaver-Burk plots. The values given are the means of two independent measurements that varied by less than 5%.

pK Value of Cysteine 40-- Cys40 is the N-terminal cysteine residue in the WCPPC active site motif of tryparedoxins (1, 2). Thioredoxins (CGPC), glutaredoxins (CPYC), and DsbA (CPHC) share similar sequences (26). In any case, the first cysteine is the nucleophile that is responsible for the reactivity of the thiol disulfide oxidoreductases. To get an insight in the molecular basis of the distinct properties of tryparedoxin, the pK value of Cys40 was determined by two independent methods.

Measurement of the Ionization State of Cys40 at 240 nm-- The absorption spectrum of reduced T. brucei tryparedoxin was monitored between 200 and 400 nm in the pH range 5-10 (Fig. 3). Because many other groups in a protein also absorb in this region, the spectra of oxidized tryparedoxin and the protein carboxamidomethylated at Cys40 were recorded for comparison. All spectra overlapped except for the region between 240 and 270 nm where reduced tryparedoxin showed a significant absorption increase with rising pH. Fig. 4 gives the epsilon  values at 240, 288, and 295 nm as a function of pH for the three protein species. Changes at 240 nm reflect ionization of thiols, at 288 nm unfolding, and at 295 nm tyrosine ionization (7). At 288 and 295 nm, the coefficients of the three proteins were almost identical and independent of the pH. The proteins were stable between pH 2.5 and 4.8 and between 5.3 and 9.6. At pH 5.0, the solubility of tryparedoxin was drastically diminished probably because of its isoelectric point, the theoretical value being 5.06. At high pH values, the epsilon 240 values of oxidized and alkylated tryparedoxin slightly increased in parallel. In contrast, the epsilon 240 value of reduced tryparedoxin yielded a sigmoidal dependence on the pH value. The concentration of the thiolate anion was calculated using an absorption coefficient epsilon 240 nm of 4.000 M-1 cm-1 (7). The value obtained is in accordance with a single thiol ionization equilibrium. The pK value was derived from the Henderson-Hasselbalch equation: Aexp = ASH + (AS- - ASH)/(1 + 10(pK-pH)), where Aexp corresponds to the A240/A280 ratio of the experimental value, ASH is the A240/A280 ratio for the fully protonated form, and AS- is the A240/A280 ratio for the fully deprotonated form (27). The approach yielded a thiol pK value of 7.2 ± 0.1. 


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  		Fig. 3.   Absorption spectra of reduced tryparedoxin at different pH values. The spectra were recorded at (solid line) pH 5.3, (dotted line) pH 6.9, (dashed line) pH 8.0, and (dashed and dotted line) pH 9.7 as described under "Experimental Procedures." 10 µM tryparedoxin in 1 mM each citrate, borate, and phosphate, and 0.2 M KCl was titrated with 0.2 M KOH, and the spectra were corrected for the resulting dilution.


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  		Fig. 4.   Titration of Cys40 of T. brucei tryparedoxin. A-C, absorption properties of reduced (open circle ), oxidized (), and carboxymethylated (black-down-triangle ) tryparedoxin as a function of pH. The spectra were recorded between 200 and 400 nm, and the extinction coefficients at 240 nm (A), 288 nm (B), and 295 nm (C) were calculated. D, extinction coefficients at 240 nm of the reduced (down-triangle) and oxidized (black-down-triangle ) thioredoxin-like mutant and the reduced (open circle ) and oxidized () glutaredoxin-like mutant. All of the measurements were carried out with 10 µM tryparedoxin in 1 mM each phosphate, citrate, and borate, and 0.2 M KCl at 25 °C. The solution was titrated from pH 5.0 to 10 with KOH and from 4.8 to 2.5 with HCl. The absorption values were corrected for the dilution.

The two central residues within the active site CXXC motif play a crucial role in determining the physicochemical properties of the thioredoxin-like proteins. The reduced and oxidized thioredoxin-like (CGPC) and glutaredoxin-like (CPYC) mutants were titrated, and the pK value was determined as described for wild type tryparedoxin. The pK value for the thioredoxin-like mutant was 7.2 ± 0.2, identical with that of the authentic tryparedoxin (Fig. 4D). In the case of the glutaredoxin-like mutant, however, the pK dropped by 3 orders of magnitude (Table II). The protein was titrated several times in steps from pH 5.2 to 7.05 and from 4.8 to 2.5, but the pK value could not be determined precisely. A typical curve is depicted in Fig. 4D. Protonation of the cysteinyl residue seemed not to be complete even at pH 2.5, although the oxidized protein also showed some variation at low pH values.

                              
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Table II
pK values of different thiols and dithiol proteins

pH Dependence of the Alkylation Rate of Tryparedoxin-- Alkylation of cysteinyl groups occurs only in the ionized thiolate anion state. Therefore, measuring the reaction rate as a function of pH can be used to determine the pK value of cysteinyl thiol groups (4, 7, 22). 50 µM of each reduced tryparedoxin and iodoacetic acid were allowed to react at pH values between 5.9 and 9.5. After different times, an aliquot of the reaction mixture was removed, the reaction was stopped by adding excess DTE, and the sample was analyzed by HPLC. The concentrations of reduced and carboxymethylated tryparedoxin were calculated from the peak area of the HPLC profile. The reaction between equal concentrations of tryparedoxin and iodoacetic acid follows the equation 1/[Tpxo - Tpxcmc] = 1/[Tpxo] + k × t, where [Tpxo] = initial concentration of tryparedoxin, [Tpxcmc] = concentration of carboxymethylated tryparedoxin, k = apparent second order rate constant, and t = time. Plots of 1/([Tpxo] - [Tpxcmc]) versus time yielded straight lines consistent with a second order reaction with a single rate constant (Fig. 5). The second order rate constants were calculated according to the equation k = [Tpxcmc]/{t × [Tpxo] × ([Tpxo] - [Tpxcmc])} (22) and plotted against pH (Fig. 6). They showed a pronounced dependence on pH between 7 and 8 but were almost constant between pH 5.9 and 6.7 as well as 8 and 8.5. As observed for DsbA, the pH dependence of the reaction followed that expected for titration of a single group but with plateau rates at high and low pH values, khi and klo, respectively (7): k = klo + (khi - klo)/(1 + 10(pK-pH)). The calculated apparent pK value of 7.2 ± 0.1 agreed well with that obtained from the absorption measurement at 240 nm. As shown in Fig. 6, a second inflection point was observed around pH 8.8. The protein species formed at high pH values is monoalkylated tryparedoxin as shown by thiol analyses and HPLC. Reduced, mono-carboxymethylated, and bis-carboxymethylated tryparedoxin (obtained under denaturing conditions) are clearly separated by HPLC, eluting after 10.7, 9.3, and 8.4 min, respectively (Fig. 7). The reaction of tryparedoxin with iodoacetamide was too fast to be followed. Even at 10 µM of both reactants, the minimum concentration allowing the subsequent HPLC analysis, alkylation of tryparedoxin, was complete in less than 5 min.


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  		Fig. 5.   Reaction of T. brucei tryparedoxin with iodoacetic acid. 50 µM reduced tryparedoxin was reacted at 25 °C with 50 µM iodoacetic acid in 10 mM each MES, MOPS, acetate, and Tris at different pH values. Shown are the data obtained at pH 6.5 (black-down-triangle ), 7.5 (open circle ), and 9.0 (). The reaction was stopped at various times by the addition of 1 volume 200 mM DTE. Carboxymethylated and unmodified tryparedoxin were separated by HPLC. Tpxo, initial concentration of tryparedoxin; Tpxcmc, concentration of carboxymethylated tryparedoxin.


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  		Fig. 6.   pH dependence of the second order rate constant (kapp) of the reaction between reduced tryparedoxin and iodoacetic acid. The second order rate constants were calculated from the reaction between 50 µM tryparedoxin and 50 µM iodoacetic acid using the equation given under "Results."


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  		Fig. 7.   Separation of reduced, mono-carboxymethylated, and bis-carboxymethylated tryparedoxin by HPLC. The protein was alkylated in the absence and presence of 6 M guanidinium chloride yielding mono-carboxymethylated (mono) and bis-carboxymethylated (bis) tryparedoxin, respectively. The proteins were separated from reduced tryparedoxin (red) by HPLC as described under "Experimental Procedures."

Determination of the Redox Potential-- The redox potential of T. brucei tryparedoxin was determined by direct protein-protein equilibration with E. coli thioredoxin. The method described by Åslund et al. (23) is convenient and has some advantages in comparison with glutathione/glutathione disulfide redox buffers. Firstly, the Eo' values reported in the literature for glutathione vary from -205 to -260 mV. Secondly, as observed with glutaredoxin, difficulties can arise if the protein forms mixed disulfides with glutathione (23).

The measurements were carried out at 25 °C and pH 7.0. Different ratios of oxidized T. brucei tryparedoxin and reduced E. coli thioredoxin and of reduced tryparedoxin and oxidized thioredoxin, respectively, were allowed to equilibrate for 4 h, and the four protein species were separated and quantified by HPLC (Fig. 8). Prior to the analysis, different amounts of each protein species were injected separately confirming that the peak areas were proportional to the protein amount. The time to achieve the redox equilibrium between the proteins was verified by incubating mixtures of oxidized tryparedoxin and reduced thioredoxin and vice versa for 2, 4, 6, and 24 h. After 4 h, a stable ratio of the four protein species had been reached that was constant after 6 h, confirming that unspecific oxidation did not occur during that time.


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  		Fig. 8.   HPLC profile of the separation of reduced and oxidized T. brucei tryparedoxin and E. coli thioredoxin. Reduced and oxidized tryparedoxin and E. coli thioredoxin were allowed to equilibrate for 4 h at 25 °C in 100 µl of 100 mM potassium phosphate, pH 7.0. The proteins were immediately separated by HPLC as described under "Experimental Procedures."

The redox potential of tryparedoxin was calculated from the following Nernst equation.
E<SUP>o′</SUP><SUB>(<UP>Tpx</UP>)</SUB>=E<SUP>o′</SUP><SUB>(<UP>Trx</UP>)</SUB>−(RT/nF)<UP>ln</UP>([<UP>Tpx<SUB>ox</SUB></UP>][<UP>Trx<SUB>red</SUB></UP>]<UP>/</UP>[<UP>Tpx<SUB>red</SUB></UP>][<UP>Trx<SUB>ox</SUB></UP>])
The standard redox potential of E. coli thioredoxin, based on the thioredoxin reductase catalyzed redox equilibrium with NADPH, is -270 mV (28). Analysis of different mixtures of oxidized and reduced tryparedoxin and thioredoxin resulted in an identical standard redox potential of -249 ± 2 mV for the parasite protein (Table III). In the case of the mutant tryparedoxins, the oxidized and reduced protein species did not well separate upon HPLC, which did not yet allow determination of their redox potential by this method.

                              
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Table III
Redox potentials of different thiols and dithiol proteins


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tryparedoxin is a highly abundant protein present in all life stages of T. brucei, the estimated cellular concentration being >100 µM. The catalytic features of tryparedoxin studied so far are intermediate between those of thioredoxins and glutaredoxins. A reaction catalyzed by glutaredoxins (thioltransferases) is reduction of dehydroascorbate by glutathione to form ascorbate, whereby the physiological significance of the reaction is still debated (24, 29). T. brucei tryparedoxin catalyzes the reaction, but the rate is even an order of magnitude lower than that with E. coli glutaredoxin. In comparison with glutathione, the spontaneous reduction of dehydroascorbate by trypanothione is 3 orders of magnitude faster (20). As shown here, the reaction is not further accelerated by tryparedoxin. Thus, as previously suggested, in trypanosomatids ascorbate seems to be kept in the reduced state by the spontaneous reaction with trypanothione.

Thioredoxins but not glutaredoxins catalyze the activation of NADP-dependent malate dehydrogenases from plants. The degree of activation and the type of saturation kinetic depend on the individual thioredoxin. Because the simple reduction of defined disulfide bridges by different thioredoxins should not induce variable cooperative effects within the dimeric NADP-malate dehydrogenase, formation of a specific complex between the two proteins has been inferred for the activation mechanism. (21). T. brucei tryparedoxin activates spinach malate dehydrogenase, but the effect is less pronounced than that observed with E. coli and T. brucei thioredoxin. The ability of tryparedoxin to activate malate dehydrogenase shows that the CGPC active site motif of thioredoxins is not a prerequisite for the reaction. This is in accordance with the finding that three thioredoxins from Arabidopsis thaliana, all containing the CPPC motif of tryparedoxin, activate NADP-malate dehydrogenase although less efficiently than chloroplastic thioredoxin (30).

The pK value of the nucleophilic cysteine and the redox potential are the main determinants for the distinct reactivities of the CXXC proteins. The pK of Cys40 of T. brucei tryparedoxin has been derived from the absorption of the thiolate anion at 240 nm as well as from the rate of carboxymethylation of the reduced protein. The value of 7.2 is comparable with that of E. coli thioredoxin (4, 22) and trypanothione (31) but strikingly different from that of other members of the thioredoxin family like yeast glutaredoxin (6) and the periplasmatic DsbA (7). The pK values of tryparedoxin and trypanothione coincide with the cellular pH of the parasites (32). This may contribute to the high reactivity of the parasite thiols because the second order rate constants for thiol-disulfide exchange reactions exhibit an optimum when the thiol pK value is equal to the pH of the surrounding solution (33).

The spectral measurements did not give evidence for a second ionization of reduced tryparedoxin at high pH. Probably ionization of Cys43 is inhibited by the presence of ionized Cys40 as has been suggested for E. coli DsbA (7). In contrast, alkylation of reduced tryparedoxin by iodoacetic acid yielded a second inflection point around pH 8.8, the origin of which is not yet clear. HPLC analysis of the reaction products and quantitative thiol determinations showed that monoalkylated tryparedoxin is formed throughout the pH range between 5 and 9.5. At high pH values where both cysteines may be deprotonated, the equal concentrations of iodoacetic acid and reduced tryparedoxin used in the experiments could lead to a competitive labeling of both residues. In E. coli thioredoxin the second inflection point around pH 9.0 was first attributed to the pK of the second cysteine (22) but was later shown to be caused by the titration of another residue increasing the pK value of the first cysteine (4). The second apparent pK value of tryparedoxin may be due to the fact that at high pH values the network of hydrogen bonds around Cys43 responsible for the lowered pK of Cys40 (17) is disturbed, yielding an increased thiol pK. Alternatively, as outlined above for E. coli thioredoxin, the second transition may result from the titration of another amino acid side chain that increases the reactivity of Cys40 with iodoacetic acid (4).

Alkylation of Cys40 in reduced T. brucei tryparedoxin by iodoacetamide is too rapid to be employed for the determination of the pH-dependent rate constants. Also the reaction with iodoacetic acid is much faster than the respective reaction of Cys32 in E. coli thioredoxin (22). In its fully ionized state, tryparedoxin was carboxymethylated with a rate constant of 22.5 M-1 s-1 (t1/2 = 15 min), which is nearly four times higher than that of E. coli thioredoxin, and this corresponds to the rates normally seen with fully ionized small thiols (34). As in E. coli DsbA (7) and thioredoxin (22), the rate decreases at low pH values but, in contrast to unstructured small peptides, does not become zero.

The pK value of the nucleophilic cysteine is significantly lower than 8.7, the pK of free cysteine, as is observed in all known members of the thioredoxin family. Low pK values of thiol groups may be due to the close proximity of positive charges. In thioredoxin-like proteins, positively charged residues are not found in the neighborhood of the redox active disulfide. Most probably, the localization of the nucleophilic cysteine at the N terminus of an alpha -helix is responsible for the lowering of the pK values (35), the positive partial charge of the helix dipol stabilizing the thiolate anion.

The crystal structures of C. fasciculata tryparedoxin revealed that the first active site cysteine is also located at the N-terminal end of an alpha -helix protruding out of the protein molecule (16, 17) and that there are no basic groups that might promote dissociation of the nucleophilic cysteine. Activation of the first cysteine is suggested to be achieved by the second cysteine, whose proton appears to be loosened by a network of hydrogen bonds (17). A fast proton shuttling between the two SH groups may ultimately help to dissociate the proton from the first cysteine. A proton sharing mechanism between the two cysteines was also described for E. coli thioredoxin (36).

Apart from the conserved localization at the N terminus of an alpha -helix, the pK value of the nucleophilic cysteine residue is determined by the two residues between the active site thiols. Replacement of these residues in E. coli thioredoxin to mimic the motif of glutaredoxin, DsbA, or protein-disulfide isomerase decreased the pK value of the nucleophilic Cys30 by up to 1.2 pH units (4). A striking shift of the pK value was observed in E. coli DsbA. Substitution of the His in the active site motif (WCPHC) by a Pro, which results in the WCPPC motif of tryparedoxins, caused an increase of the pK of Cys30 by more than 3 orders of magnitude from 3.5 to 6.73 (27). In T. brucei tryparedoxin, changing CPPC into the CGPC motif of thioredoxin did not affect the pK value. This is not surprising because the pK values of the authentic proteins are very similar. In contrast, introduction of the CPYC motif of glutaredoxin lowered the pK of Cys40 by about 3 orders of magnitude. Thus, replacement of the second proline by a tyrosine in tryparedoxin was sufficient to yield a pK value comparable with that of authentic glutaredoxins.

The midpoint potentials among the different members of the thioredoxin family range from -124 mV for E. coli DsbA to -270 mV for E. coli thioredoxin. The Eo' = -249 mV of T. brucei tryparedoxin classifies the parasite protein as a reducing representative, but it should be kept in mind that the in vivo functions of the thiol disulfide oxidoreductases may depend on the redox environment. For E. coli thioredoxin I it was shown that changing the location from cytoplasm to periplasm alters the function of the protein from a reductant to an oxidant (37). The redox potential of tryparedoxin is intermediate between those of E. coli glutaredoxin (-233 mV) (23) and thioredoxin. The physiological electron donor for tryparedoxin is the dithiol trypanothione, which has a comparable redox potential of -242 mV (8). Thus, the concentrations of tryparedoxin and trypanothione are probably the predominant factors in determining the equilibrium state of the system. As shown recently, the activity of tryparedoxin is strongly influenced by the trypanothione/trypanothione disulfide ratio. In the presence of 1 mM reduced trypanothione, the IC50 value of tryparedoxin for trypanothione disulfide is 50 µM (14). A respective interdependence has been reported for E. coli thioredoxin that is strongly inhibited by a lowered 2GSH/GSSG ratio (38). The fact that in the trypanothione/tryparedoxin as well as the glutathione/glutaredoxin systems the reaction partners have very similar redox potentials may facilitate the control of the dithiol/disulfide ratio in the proteins by the (di)thiol/disulfide ratio of the respective nonprotein (di)thiol (14, 38).

The intracellular concentration of tryparedoxin is higher than 100 µM and thus in the same order of magnitude as trypanothione (8). Together with the very similar redox potentials, this indicates that the physiological equilibrium of tryparedoxin/trypanothione is near 1, which allows the couple to respond readily to small changes in the redox milieu. In mammalian cells, the redox potential of the cytosol has been estimated to be around -235 mV (39). When assuming that this is also true for trypanosomes, the trypanothione/tryparedoxin couple is probably the most important factor for determining the cytosolic redox potential of the parasites.

    ACKNOWLEDGEMENTS

Dr. Hartmut Follmann (University of Kassel, Germany) is kindly acknowledged for a gift of spinach malate dehydrogenase. We thank Dr. R. Heiner Schirmer (Biochemie-Zentrum, Universität Heidelberg, Germany) for a sample of human thioredoxin reductase and for many helpful discussions. We are indebted to Dr. Manuela López de la Paz (EMBL Heidelberg) for help with recording and interpretation of the CD spectra.

    FOOTNOTES

* Our work is supported by Deutsche Forschungsgemeinschaft Grant SFB 544 "Control of Tropical Infectious Diseases."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.

Dagger To whom correspondence should be addressed: Biochemie-Zentrum Heidelberg, Ruprecht-Karls-Universität, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Tel.: 49-6221-54-41-87; Fax: 49-6221-54-55-86; E-mail: krauth-siegel@urz.uni-heidelberg.de.

Published, JBC Papers in Press, February 26, 2002, DOI 10.1074/jbc.M112115200

    ABBREVIATIONS

The abbreviations used are: T(SH)2, trypanothione [N1,N8-bis(glutathionyl)spermidine]; DTE, dithioerythritol; HPLC, high pressure liquid chromatography; MES, morpholinoethane sulfonic acid; MOPS, morpholinopropane sulfonic acid; Tpx, tryparedoxin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Gommel, D. U., Nogoceke, E., Morr, M., Kiess, M., Kalisz, H. M., and Flohé, L. (1997) Eur. J. Biochem. 248, 913-918[Medline] [Order article via Infotrieve]
2. Lüdemann, H., Dormeyer, M., Sticherling, C., Stallmann, D., Follmann, H., and Krauth-Siegel, R. L. (1998) FEBS Lett. 431, 381-385[CrossRef][Medline] [Order article via Infotrieve]
3. Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237-271[CrossRef][Medline] [Order article via Infotrieve]
4. Mössner, E., Huber-Wunderlich, M., and Glockshuber, R. (1998) Protein Sci. 7, 1233-1244[Abstract]
5. Holmgren, A. (1989) J. Biol. Chem. 264, 13963-13966[Free Full Text]
6. Gan, Z.-R., Sardana, M. K., Jacobs, J. W., and Polokoff, M. A. (1990) Arch. Biochem. Biophys. 282, 110-115[CrossRef][Medline] [Order article via Infotrieve]
7. Nelson, J. W., and Creighton, T. E. (1994) Biochemistry 33, 5974-5983[CrossRef][Medline] [Order article via Infotrieve]
8. Fairlamb, A. H., and Cerami, A. (1992) Annu. Rev. Microbiol. 46, 695-729[CrossRef][Medline] [Order article via Infotrieve]
9. Krauth-Siegel, R. L., and Coombs, G. (1999) Parasitol. Today 15, 404-409[CrossRef][Medline] [Order article via Infotrieve]
10. Flohé, L., Hecht, H. J., and Steinert, P. (1999) Free Radic. Biol. Med. 27, 966-984[CrossRef][Medline] [Order article via Infotrieve]
11. Lopez, J. A., Carvalho, T. U., de Souza, W., Flohé, L., Guerrero, S. A., Montemartini, M., Kalisz, H. M., Nogoceke, E., Singh, M., Alves, M. J. M., and Colli, W. (2000) Free Radic. Biol. Med. 28, 767-772[CrossRef][Medline] [Order article via Infotrieve]
12. Ivens, A. C., Lewis, S. M., Bagherzadeh, A., Zhang, L., Chan, H. M., and Smith, D. F. (1998) Genome Res. 8, 135-145[Abstract/Free Full Text]
13. Nogoceke, E., Gommel, D. U., Kiebeta , M., Kalisz, H. M., and Flohé, L. (1997) Biol. Chem. 378, 827-836[Medline] [Order article via Infotrieve]
14. Dormeyer, M., Reckenfelderbäumer, N., Lüdemann, H., and Krauth-Siegel, R. L. (2001) J. Biol. Chem. 276, 10602-10606[Abstract/Free Full Text]
15. Reckenfelderbäumer, N., Lüdemann, H., Schmidt, H., Steverding, D., and Krauth-Siegel, R. L. (2000) J. Biol. Chem. 275, 7547-7552[Abstract/Free Full Text]
16. Alphey, M. S., Leonard, G. A., Gourley, D. G., Tetaud, E., Fairlamb, A. H., and Hunter, W. N. (1999) J. Biol. Chem. 274, 25613-25622[Abstract/Free Full Text]
17. Hofmann, B., Budde, H., Bruns, K., Guerrero, S. A., Kalisz, H. M., Menge, U., Montemartini, M., Nogoceke, E., Steinert, P., Wissing, J. B., Flohé, L., and Hecht, H.-J. (2001) Biol. Chem. 382, 459-471[CrossRef][Medline] [Order article via Infotrieve]
18. Sullivan, F. X., and Walsh, C. T. (1991) Mol. Biochem. Parasitol. 44, 145-147[CrossRef][Medline] [Order article via Infotrieve]
19. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77[CrossRef][Medline] [Order article via Infotrieve]
20. Krauth-Siegel, R. L., and Lüdemann, H. (1996) Mol. Biochem. Parasitol. 80, 203-208[CrossRef][Medline] [Order article via Infotrieve]
21. Braun, H., Lichter, A., and Häberlein, I. (1996) Eur. J. Biochem. 240, 781-788[Medline] [Order article via Infotrieve]
22. Kallis, G.-B., and Holmgren, A. (1980) J. Biol. Chem. 255, 10261-10265[Abstract/Free Full Text]
23. Åslund, F., Berndt, K. D., and Holmgren, A. (1997) J. Biol. Chem. 272, 30780-30786[Abstract/Free Full Text]
24. Washburn, M. P., and Wells, W. W. (1999) Biochemistry 38, 268-274[CrossRef][Medline] [Order article via Infotrieve]
25. May, J. M., Mendiratta, S., Hill, K. E., and Burk, R. F. (1997) J. Biol. Chem. 272, 22607-22610[Abstract/Free Full Text]
26. Chivers, P. T., Prehoda, K. E., and Raines, R. T. (1997) Biochemistry 36, 4061-4066[CrossRef][Medline] [Order article via Infotrieve]
27. Grauschopf, U., Winther, J. R., Korber, P., Zander, T., Dallinger, P., and Bardwell, J. C. A. (1995) Cell 83, 947-955[CrossRef][Medline] [Order article via Infotrieve]
28. Krause, G., Lundström, J., Lopez-Barea, J.,., Pueyo de la Cuesta, C., and Holmgren, A. (1991) J. Biol. Chem. 266, 9494-9500[Abstract/Free Full Text]
29. Winkler, B. S., Orselli, S. M., and Rex, T. S. (1994) Free Radic. Biol. Med. 17, 333-349[CrossRef][Medline] [Order article via Infotrieve]
30. Rivera-Madrid, R., Mestres, D., Marinho, P., Jacquot, J. P., Decottignies, P., Miginiac-Maslow, M., and Meyer, Y. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5620-5624[Abstract/Free Full Text]
31. Moutiez, M., Meziane-Cherif, D., Aumercier, M., Sergheraert, C., and Tartar, A. (1994) Chem. Pharm. Bull. 42, 2641-2644
32. Fraser-L'Hostis, C., Defrise-Quertin, F., Coral, D., and Deshusses, J. (1997) Biol. Chem. 378, 1039-1046[Medline] [Order article via Infotrieve]
33. Gilbert, H. F. (1990) Adv. Enzymol. Relat. Areas Mol. Biol. 63, 69-172[Medline] [Order article via Infotrieve]
34. Snyder, G. H. (1987) Biochemistry 26, 688-694[CrossRef][Medline] [Order article via Infotrieve]
35. Kortemme, T., and Creighton, T. E. (1995) J. Mol. Biol. 253, 799-812[CrossRef][Medline] [Order article via Infotrieve]
36. Jeng, M.-F., Holmgren, A., and Dyson, H. J. (1995) Biochemistry 34, 10101-10105[CrossRef][Medline] [Order article via Infotrieve]
37. Debarbieux, L., and Beckwith, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10751-10756[Abstract/Free Full Text]
38. Holmgren, A. (1979) J. Biol. Chem. 254, 3672-3678[Free Full Text]
39. Keese, M. A., Saffrich, R., Dandekar, T., Becker, K., and Schirmer, R. H. (1999) FEBS Lett. 447, 135-138[CrossRef][Medline] [Order article via Infotrieve]

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