Catalytic Properties, Thiol p K Value, and Redox Potential of Trypanosoma brucei Tryparedoxin
2002; Elsevier BV; Volume: 277; Issue: 20 Linguagem: Inglês
10.1074/jbc.m112115200
ISSN1083-351X
AutoresNina Reckenfelderbäumer, R. Luise Krauth‐Siegel,
Tópico(s)Synthesis and biological activity
ResumoThe 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 p K 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 p K value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the p K dropped to ≤4.0. The fact that the p K value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions. 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 p K 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 p K value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the p K dropped to ≤4.0. The fact that the p K value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions. Tryparedoxins are 16-kDa dithiol proteins that belong to the large family of thioredoxin-like thiol-disulfide oxidoreductases, all of which share a C XXC active site motif by low overall sequence similarities. The redox active motif reads CPPC in tryparedoxins (1Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar,2Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar), CGPC in thioredoxins (3Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 4Mössner E. Huber-Wunderlich M. Glockshuber R. Protein Sci. 1998; 7: 1233-1244Crossref PubMed Scopus (157) Google Scholar), CPYC in glutaredoxins (5Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 6Gan Z.-R. Sardana M.K. Jacobs J.W. Polokoff M.A. Arch. Biochem. Biophys. 1990; 282: 110-115Crossref PubMed Scopus (43) Google Scholar), CGHC in eukaryotic protein-disulfide isomerases, and CPHC in the bacterial periplasmatic protein thiol:disulfide oxidoreductase DsbA (7Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-5983Crossref PubMed Scopus (225) Google Scholar). 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 (8Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (691) Google Scholar, 9Krauth-Siegel R.L. Coombs G. Parasitol. Today. 1999; 15: 404-409Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10Flohé L. Hecht H.J. Steinert P. Free Radic. Biol. Med. 1999; 27: 966-984Crossref PubMed Scopus (184) Google Scholar). 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 (9Krauth-Siegel R.L. Coombs G. Parasitol. Today. 1999; 15: 404-409Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Initially tryparedoxin was isolated from the insect parasite Crithidia fasciculata (1Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar), but the protein occurs also in the pathogenic trypanosomatids T. brucei (2Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar), T. cruzi (11Lopez 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. Colli W. Free Radic. Biol. Med. 2000; 28: 767-772Crossref PubMed Scopus (52) Google Scholar), and L. major (12Ivens A.C. Lewis S.M. Bagherzadeh A. Zhang L. Chan H.M. Smith D.F. Genome Res. 1998; 8: 135-145Crossref PubMed Scopus (71) Google Scholar). 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 (1Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar, 13Nogoceke E. Gommel D.U. Kieβ M. Kalisz H.M. Flohé L. Biol. Chem. 1997; 378: 827-836Crossref PubMed Scopus (264) Google Scholar). We have cloned and overexpressed the gene encoding tryparedoxin from T. brucei (2Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). The recombinant protein catalyzes the trypanothione-dependent reduction of ribonucleotide reductase during the parasite synthesis of DNA precursors (14Dormeyer M. Reckenfelderbäumer N. Lüdemann H. Krauth-Siegel R.L. J. Biol. Chem. 2001; 276: 10602-10606Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In the latter reaction, tryparedoxin thus can replace the well known thioredoxin and/or glutaredoxin systems in other organisms (5Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). T. brucei also possesses a classical thioredoxin, but its concentration is unusually low (15Reckenfelderbäumer N. Lüdemann H. Schmidt H. Steverding D. Krauth-Siegel R.L. J. Biol. Chem. 2000; 275: 7547-7552Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 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 (2Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). 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 β-sheet surrounded by four α-helices (16Alphey M.S. Leonard G.A. Gourley D.G. Tetaud E. Fairlamb A.H. Hunter W.N. J. Biol. Chem. 1999; 274: 25613-25622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Hofmann B. Budde H. Bruns K. Guerrero S.A. Kalisz H.M. Menge U. Montemartini M. Nogoceke E. Steinert P. Wissing J.B. Flohé L. Hecht H.-J. Biol. Chem. 2001; 382: 459-471Crossref PubMed Scopus (54) Google Scholar). In particular the active site motif Cys40-Pro41-Pro42-Cys43is 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 p K value of the first cysteine residue in the C XXC 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 p K 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. Trypanothione disulfide was purchased from Bachem. The reduced form, trypanothione, T(SH)2 1The abbreviations used are: T(SH)2trypanothione [N1,N8-bis(glutathionyl)spermidine]DTEdithioerythritolHPLChigh pressure liquid chromatographyMESmorpholinoethane sulfonic acidMOPSmorpholinopropane sulfonic acidTpxtryparedoxin was prepared by NaBH4 reduction as described (14Dormeyer M. Reckenfelderbäumer N. Lüdemann H. Krauth-Siegel R.L. J. Biol. Chem. 2001; 276: 10602-10606Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). 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 (2Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar), T. cruzi trypanothione reductase (18Sullivan F.X. Walsh C.T. Mol. Biochem. Parasitol. 1991; 44: 145-147Crossref PubMed Scopus (102) Google Scholar), and T. brucei thioredoxin (15Reckenfelderbäumer N. Lüdemann H. Schmidt H. Steverding D. Krauth-Siegel R.L. J. Biol. Chem. 2000; 275: 7547-7552Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) 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. trypanothione [N1,N8-bis(glutathionyl)spermidine] dithioerythritol high pressure liquid chromatography morpholinoethane sulfonic acid morpholinopropane sulfonic acid tryparedoxin In a total volume of 400 μl, 200 μmT. 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 (19Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21618) Google Scholar). Carboxamidomethylation of tryparedoxin was carried out as described (14Dormeyer M. Reckenfelderbäumer N. Lüdemann H. Krauth-Siegel R.L. J. Biol. Chem. 2001; 276: 10602-10606Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Carboxymethylated tryparedoxin was prepared by incubating 50 μmtryparedoxin 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. 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 μmT. brucei tryparedoxin,E. coli glutaredoxin, and thioredoxin, respectively. Formation of ascorbate was followed at 25 °C (ε265 = 14,000 m−1 cm−1) in a Beckman DU-65 spectrophotometer (20Krauth-Siegel R.L. Lüdemann H. Mol. Biochem. Parasitol. 1996; 80: 203-208Crossref PubMed Scopus (53) Google Scholar). The protein-mediated activity was corrected for the spontaneous reaction rate. Reduction of 100 μmdehydroascorbate 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. In a total volume of 80 μl, the reaction mixtures contained 50 and 100 μm dehydroascorbate, respectively, in 100 mmpotassium 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 μmT. brucei tryparedoxin. 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 mmpotassium 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 μmE. 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 (21Braun H. Lichter A. Häberlein I. Eur. J. Biochem. 1996; 240: 781-788Crossref PubMed Scopus (13) Google Scholar). 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 (2Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). 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. 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. The pH-dependent ionization of the cysteine 40 thiol was monitored by the absorbance of the thiolate anion at 240 nm (7Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-5983Crossref PubMed Scopus (225) Google Scholar). 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 μmtryparedoxin, 1 mm each citrate, borate, and phosphate, 0.2m 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. 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 (7Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-5983Crossref PubMed Scopus (225) Google Scholar, 22Kallis G.-B. Holmgren A. J. Biol. Chem. 1980; 255: 10261-10265Abstract Full Text PDF PubMed Google Scholar). 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. The redox potential of T. brucei tryparedoxin was determined by direct protein-protein redox equilibration (23Åslund F. Berndt K.D. Holmgren A. J. Biol. Chem. 1997; 272: 30780-30786Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). 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. In vitro activity of glutaredoxins (thioltransferases) is the ability to catalyze the reduction of dehydroascorbate by glutathione (24Washburn M.P. Wells W.W. Biochemistry. 1999; 38: 268-274Crossref PubMed Scopus (53) Google Scholar). Thioredoxins do not catalyze the reaction whereby a recent report shows that the mammalian thioredoxin reductase/thioredoxin system can reduce dehydroascorbate (25May J.M. Mendiratta S. Hill K.E. Burk R.F. J. Biol. Chem. 1997; 272: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). 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 (20Krauth-Siegel R.L. Lüdemann H. Mol. Biochem. Parasitol. 1996; 80: 203-208Crossref PubMed Scopus (53) Google Scholar). The reaction mixtures contained 100 μmdehydroascorbate, 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 μmE. 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 1300m−1 min−1 (20Krauth-Siegel R.L. Lüdemann H. Mol. Biochem. Parasitol. 1996; 80: 203-208Crossref PubMed Scopus (53) Google Scholar). 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. 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 (21Braun H. Lichter A. Häberlein I. Eur. J. Biochem. 1996; 240: 781-788Crossref PubMed Scopus (13) Google Scholar). 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 (21Braun H. Lichter A. Häberlein I. Eur. J. Biochem. 1996; 240: 781-788Crossref PubMed Scopus (13) Google Scholar). 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 (21Braun H. Lichter A. Häberlein I. Eur. J. Biochem. 1996; 240: 781-788Crossref PubMed Scopus (13) Google Scholar). 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 (2Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). 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 α-helices and β-sheets in the three protein species. Mammalian thioredoxin reductases show a broad substrate specificity accepting thioredoxins from different species. T. brucei tryparedoxin is reduced by human thioredoxin reductase with a Kmvalue 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 (15Reckenfelderbäumer N. Lüdemann H. Schmidt H. Steverding D. Krauth-Siegel R.L. J. Biol. Chem. 2000; 275: 7547-7552Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 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.Table IKinetic parameters for the reduction of different T. brucei tryparedoxin species by human thioredoxin reductaseTryparedoxin speciesActive site motifKmVmaxRelative efficiencyμM%%Wild typeCPPC43.5100100Thioredoxin mutantCGPC17.42870Glutaredoxin mutantCPYC20026858The 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%. Open table in a new tab 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%. Cys40 is the N-terminal cysteine residue in the WCPPC active site motif of tryparedoxins (1Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar,2Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). Thioredoxins (CGPC), glutaredoxins (CPYC), and DsbA (CPHC) share similar sequences (26Chivers P.T. Prehoda K.E. Raines R.T. Biochemistry. 1997; 36: 4061-4066Crossref PubMed Scopus (235) Google Scholar). 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 p K value of Cys40 was determined by two independent methods. 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 Cys40were 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 ε 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 (7Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-5983Crossref PubMed Scopus (225) Google Scholar). 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 ε240 values of oxidized and alkylated tryparedoxin slightly increased in parallel. In contrast, the ε240 value of reduced tryparedoxin yielded a sigmoidal dependence on the pH value. The concentration of the thiolate anion was calculated using an absorption coefficient ε240 nm of 4.000 m−1 cm−1 (7Nelson J.W. Creighton T.E. Biochemistry. 1994; 33: 5974-59
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