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W1964119106 abstract "Glutaredoxins are small proteins with a conserved active site (-CXX(C/444)-) and thioredoxin fold. These thiol disulfide oxidoreductases catalyze disulfide reductions, preferring GSH-mixed disulfides as substrates. We have developed a new real-time fluorescence-based method for measuring the deglutathionylation activity of glutaredoxins using a glutathionylated peptide as a substrate. Mass spectrometric analysis showed that the only intermediate in the reaction is the glutaredoxin-GSH mixed disulfide. This specificity was solely dependent on the unusual γ-linkage present in glutathione. The deglutathionylation activity of both wild-type Escherichia coli glutaredoxin and the C14S mutant was competitively inhibited by oxidized glutathione, with Ki values similar to the Km values for the glutathionylated peptide substrate, implying that glutaredoxin primarily recognizes the substrate via the glutathione moiety. In addition, wild-type glutaredoxin showed a sigmoidal dependence on GSH concentrations, the activity being significantly decreased at low GSH concentrations. Thus, under oxidative stress conditions, where the ratio of GSH/GSSG is decreased, the activity of glutaredoxin is dramatically reduced, and it will only have significant deglutathionylation activity once the oxidative stress has been removed. Different members of the protein disulfide isomerases (PDI) family showed lower activity levels when compared with glutaredoxins; however, their deglutathionylation activities were comparable with their oxidase activities. Furthermore, in contrast to the glutaredoxin-GSH mixed disulfide intermediate, the only intermediate in the PDI-catalyzed reaction was PDI peptide mixed disulfide. Glutaredoxins are small proteins with a conserved active site (-CXX(C/444)-) and thioredoxin fold. These thiol disulfide oxidoreductases catalyze disulfide reductions, preferring GSH-mixed disulfides as substrates. We have developed a new real-time fluorescence-based method for measuring the deglutathionylation activity of glutaredoxins using a glutathionylated peptide as a substrate. Mass spectrometric analysis showed that the only intermediate in the reaction is the glutaredoxin-GSH mixed disulfide. This specificity was solely dependent on the unusual γ-linkage present in glutathione. The deglutathionylation activity of both wild-type Escherichia coli glutaredoxin and the C14S mutant was competitively inhibited by oxidized glutathione, with Ki values similar to the Km values for the glutathionylated peptide substrate, implying that glutaredoxin primarily recognizes the substrate via the glutathione moiety. In addition, wild-type glutaredoxin showed a sigmoidal dependence on GSH concentrations, the activity being significantly decreased at low GSH concentrations. Thus, under oxidative stress conditions, where the ratio of GSH/GSSG is decreased, the activity of glutaredoxin is dramatically reduced, and it will only have significant deglutathionylation activity once the oxidative stress has been removed. Different members of the protein disulfide isomerases (PDI) family showed lower activity levels when compared with glutaredoxins; however, their deglutathionylation activities were comparable with their oxidase activities. Furthermore, in contrast to the glutaredoxin-GSH mixed disulfide intermediate, the only intermediate in the PDI-catalyzed reaction was PDI peptide mixed disulfide. Glutaredoxins are small thiol disulfide oxidoreductases, with a conserved active site sequence -CXXC- or -CXXS- and a GSH recognition site (1Holmgren A. J. Biol. Chem. 1979; 254: 3664-3671Abstract Full Text PDF PubMed Google Scholar, 2Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar, 3Höög J.O. Jörnvall H. Holmgren A. Carlquist M. Persson M. Eur. J. Biochem. 1983; 136: 223-232Crossref PubMed Scopus (80) Google Scholar, 4Nordstrand K. Sandström A. Åslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 2000; 303: 423-432Crossref PubMed Scopus (38) Google Scholar), that belong to the thioredoxin superfamily (5Xia T.-H. Bushweller J.H. Sodano P. Billeter M. Björnberg O. Holmgren A. Wüthrich K. Protein Sci. 1992; 1: 310-321Crossref PubMed Scopus (112) Google Scholar, 6Martin J.L. Structure (Lond.). 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar). Glutaredoxins use the reducing power of GSH to catalyze disulfide reductions (2Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar). They preferentially catalyze reductions of GSH-mixed disulfides but have also been suggested to function as general protein disulfide reductants (7Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar). Multiple glutaredoxins are known in different organisms (8Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (163) Google Scholar, 9Gvakharia B.O. Hanson E. Koonin E.K. Mathews C.K. J. Biol. Chem. 1996; 271: 15307-15310Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 10Rodríguez-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (263) Google Scholar, 11Lundberg M. Johansson C. Chandra J. Enoksson M. Jacobsson G. Ljung J. Johansson M. Holmgren A. J. Biol. Chem. 2001; 276: 26269-26275Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 12Gladyshev V.N. Liu A. Novoselov S.V. Krysan K. Sun Q.-A. Kryukov V.M. Kryukov G.V. Lou M.F. J. Biol. Chem. 2001; 276: 30374-30380Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), but the role of these proteins in cells and in different cell organelles is still unclear. Glutaredoxins are thought to have a primary role in defense against oxidative stress (13Collinson E.J. Grant C.M. J. Biol. Chem. 2003; 278: 22492-22497Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 14Starke D.W. Chock P.B. Mieyal J.J. J. Biol. Chem. 2003; 278: 14607-14613Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 15Murata H. Ihara Y. Nakamura H. Yodoi J. Sumikawa K. Kondo T. J. Biol. Chem. 2003; 278: 50226-50233Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 16Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), but they have been suggested to participate in other functions, such as cellular differentiation (17Takashima Y. Hirota K. Nakamura H. Nakamura T. Akiyama K. Cheng F.S. Maeda M. Yodoi J. Immunol. Lett. 1999; 68: 397-401Crossref PubMed Scopus (44) Google Scholar), redox regulation of signal transduction (14Starke D.W. Chock P.B. Mieyal J.J. J. Biol. Chem. 2003; 278: 14607-14613Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 18Bandyopadhyay S. Starke D.W. Mieyal J.J. Gronostajski R.M. J. Biol. Chem. 1998; 273: 392-397Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and prevention of apoptosis (15Murata H. Ihara Y. Nakamura H. Yodoi J. Sumikawa K. Kondo T. J. Biol. Chem. 2003; 278: 50226-50233Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar).GSH is the primary cellular low molecular weight reductant, and accordingly, there are high levels of GSH in most cells, the cellular levels ranging from 3.5 to 6.6 mm in Escherichia coli (19Apontoweil P. Berends W. Biochim. Biophys. Acta. 1975; 399: 1-9Crossref PubMed Scopus (75) Google Scholar) and from 1 to 8 mm in mammalian cells (20Griffith O.W. Free Radic. Biol. Med. 1999; 27: 922-935Crossref PubMed Scopus (960) Google Scholar). Glutathione may also be found in oxidative cellular compartments such as the endoplasmic reticulum as a disulfide linked dipeptide, GSSG, or as protein-GSH mixed disulfides (21Bass R. Ruddock L.W. Klappa P. Freedman R.B. J. Biol. Chem. 2004; 279: 5257-5262Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 22Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1571) Google Scholar). Protein-GSH mixed disulfides have also been observed to accumulate in other cellular compartments, e.g. the cytoplasm, during oxidative stress (23Cotgreave I.A. Gerdes R.G. Biochem. Biophys. Res. Commun. 1998; 242: 1-9Crossref PubMed Scopus (430) Google Scholar). The primary function of glutaredoxins is thought to be the reduction of protein-GSH mixed disulfides, liberating the native functional protein (24Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (322) Google Scholar).Glutaredoxins exist in monothiol (CXXS) and dithiol (CXXC) active site variants. The N-terminal active site cysteine is essential and sufficient for the reduction of GSH-mixed disulfides (25Bushweller J.H. Åslund F. Wüthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (203) Google Scholar, 26Yang y. Wells W.W. J. Biol. Chem. 1991; 266: 12759-12765Abstract Full Text PDF PubMed Google Scholar). The monothiol mechanism has been suggested to proceed via a pathway where the thiolate of Grx 2The abbreviations used are: Grx, glutaredoxin; HED, β-hydroxyethyl disulfide; PDI, protein disulfide isomerases. 2The abbreviations used are: Grx, glutaredoxin; HED, β-hydroxyethyl disulfide; PDI, protein disulfide isomerases. initiates a nucleophilic attack on the mixed disulfide between protein thiol and GSH, leading to a formation of a new disulfide between Grx and GSH and the release of a protein substrate in the reduced form (Fig. 1, reaction 1) (24Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (322) Google Scholar, 27Fernandes A.P. Holmgren A. Antioxid. Redox. Signal. 2004; 6: 63-74Crossref PubMed Scopus (533) Google Scholar, 28Srinivasan U. Mieyal P.A. Mieyal J.J. Biochemistry. 1997; 36: 3199-3206Crossref PubMed Scopus (108) Google Scholar, 29Lundström-Ljung J. Vlamis-Gardikas A. Åslund F. Holmgren A. FEBS Lett. 1999; 443: 85-88Crossref PubMed Scopus (25) Google Scholar, 30Gravina S.A. Mieyal J.J. Biochemistry. 1993; 32: 3368-3376Crossref PubMed Scopus (275) Google Scholar). The mixed disulfide between Grx and GSH can be reduced by GSH through a nucleophilic attack on the disulfide to form GSSG and reduced Grx as the final products (Fig. 1, reaction 2) (25Bushweller J.H. Åslund F. Wüthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (203) Google Scholar, 27Fernandes A.P. Holmgren A. Antioxid. Redox. Signal. 2004; 6: 63-74Crossref PubMed Scopus (533) Google Scholar, 28Srinivasan U. Mieyal P.A. Mieyal J.J. Biochemistry. 1997; 36: 3199-3206Crossref PubMed Scopus (108) Google Scholar). The dithiol mechanism proceeds via the same pathway except that there is partitioning at the Grx-GSH mixed disulfide state to form oxidized Grx (Fig. 1, reaction 3). This can be retrieved to the functional pathway by reduction by GSH (Fig. 1, reaction 4) (31Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (129) Google Scholar).Grx activity is often measured using an artificial nonspecific substrate β-hydroxyethyl disulfide (HED) in a spectrophotometric coupled assay (1Holmgren A. J. Biol. Chem. 1979; 254: 3664-3671Abstract Full Text PDF PubMed Google Scholar). Less frequently, substrates such as cysteine-glutathione mixed disulfide (31Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (129) Google Scholar) or glutathionylated protein substrates are used (28Srinivasan U. Mieyal P.A. Mieyal J.J. Biochemistry. 1997; 36: 3199-3206Crossref PubMed Scopus (108) Google Scholar, 29Lundström-Ljung J. Vlamis-Gardikas A. Åslund F. Holmgren A. FEBS Lett. 1999; 443: 85-88Crossref PubMed Scopus (25) Google Scholar). Here we report the development of a new real-time method for measuring the deglutathionylation activity of glutaredoxins. The assay is based on a homogeneous glutathionylated peptide and measures the actual formation of deglutathionylated product rather than indirectly the consumption of NADPH as in the traditional HED assay. This method offers new insights into the mechanism of action of the glutaredoxins and allows the glutaredoxin-like activity of other thioredoxin superfamily members, such as protein disulfide isomerases (PDI), to be determined. We also demonstrate that the reaction intermediate in the deglutathionylation reaction is exclusively Grx-GSH mixed, disulfide confirming a previous report (31Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (129) Google Scholar), and that it is dependent on the unusual γ-linkage present in GSH.EXPERIMENTAL PROCEDURESProtein Expression and Purification—All constructs used in the experiments were cloned into an expression vector generated previously (32Alanen H.I. Salo K.E. Pekkala M. Siekkinen H.M. Pirneskoski A. Ruddock L.W. Antioxid. Redox. Signal. 2003; 5: 367-374Crossref PubMed Scopus (70) Google Scholar), which incorporates an N-terminal His tag to the cloned gene. E. coli Grx1 was cloned from E. coli strain XL1-Blue, and yeast Grx1 was cloned from Saccharomyces cerevisiae strain W303. Site-directed mutagenesis was performed according to the instructions of the QuikChange™ kit (Strategene, La Jolla, CA). All plasmids were checked for correctness by sequencing. Proteins were expressed in E. coli strains BL21 (DE3) pLysS or Rosetta-gami and purified by immobilized metal affinity chromatography and ion exchange chromatography as described for the a domain of PDI (33Lappi A.-K. Lensink M.F. Alanen H.I. Salo K.E.H. Lobell M. Juffer A.H. Ruddock L.W. J. Mol. Biol. 2004; 335: 283-295Crossref PubMed Scopus (115) Google Scholar). Pure fractions, as determined by Coomassie Brilliant Blue-stained SDS-PAGE, were combined and buffer-exchanged into 20 mm sodium phosphate buffer, pH 7.3, and stored frozen. The concentration of each protein was determined spectrophotometrically using a calculated molar absorption coefficient. All purified proteins were analyzed for authenticity by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.Assay for Determining the Deglutathionylation Activity—The glutathionylated substrate peptide SQLWC(glutathione)LSN was ordered from The Biomolecular Science Facility, Department of Biosciences, University of Kent (Canterbury, Kent, UK). Fluorescence measurements were performed with a PerkinElmer Life Sciences LS50B spectrometer using a 315-μl cuvette. Assays were carried out in McIlvaine buffer (0.2 m disodium hydrogen phosphate, 0.1 m citric acid) at pH 4.5–7.5, including appropriate amounts of GSH (0–4 mm), NADPH (50 μm), glutathione reductase (0 or 20 nm) (all from Sigma), EDTA (1 mm), substrate peptide (0–20 μm), and the enzyme of interest (0–200 nm). To prevent loss of Grx due to adsorption on the walls of plastic tubes and the cuvette, dilutions of Grx and glutathione reductase were made into solutions containing bovine serum albumin, the final concentration of bovine serum albumin in the assay being 1 μg/ml. All measurements were done at 25 °C, excitation 280 nm, emission 356 nm, and slit widths 5 nm.Analysis of Fluorescence Data—The fluorescence data were analyzed with Igor Pro 3.14 (Wavemetrics Inc., Lake Oswego, OR). Initial rates of reaction were determined from linear fits over 20 units of change in fluorescence signal or other applicable linear part of the curve and correlating it to the total change in fluorescence observed during the reaction. The first few data points were ignored due to extra noise being observed during the first few seconds. The corresponding non-catalyzed background reaction rates (i.e. in the absence of Grx) were subtracted from the catalyzed rates. The change in fluorescence during the deglutathionylation reaction was proportional to the substrate concentration and otherwise independent of the reaction conditions except when glutathione reductase was omitted. In the absence of glutathione reductase, the overall change in fluorescence was up to 25% smaller, presumably due to the presence of an equilibrium between the deglutathionylation and glutathionylation reactions.Assay for Determining the Glutathionylation Activity—The reduced substrate peptide SQLWCLSN was ordered from The Biomolecular Science Facility, Department of Biosciences, University of Kent. Fluorescence measurements were performed in McIlvaine buffer (0.2 m disodium hydrogen phosphate, 0.1 m citric acid) at pH 7.0, including GSSG (5 mm), bovine serum albumin (1 μg/ml), EDTA (1 mm), reduced substrate peptide (5 μm), and the enzyme of interest (0–200 nm). Measurements were done at 25 °C, excitation 280 nm, emission 356 nm, and slit widths 5 nm.Synthesis of Alternative Substrate—The reduced substrate peptide (100 μm) was incubated with ECG, QCG, or paraECG (1 mm) in 0.1 m sodium phosphate buffer at pH 8 for 1 h. Peptide-ECG, -QCG, and -paraECG mixed disulfides were purified by reverse phase-high pressure liquid chromatography using SOURCE 5RPC ST 4.6/150 column (Amersham Biosciences, Uppsala, Sweden). The peptides were eluted from the column with a linear gradient from buffer A (0.1% trifluoroacetic acid) to 100% buffer B (90% acetonitrile, 0.1% trifluoroacetic acid) over 10 column volumes. The peptides were dried by speed vacuum and resuspended into 20 mm phosphate buffer, pH 7.3.Analysis of Reaction Intermediates by Mass Spectrometry—E. coli Grx1 wild type and C14S mutant and PDI a C39S mutant were used in trapping experiments. The protein was reduced with 1 mm dithiothreitol for 30 min at room temperature. Excess of dithiothreitol was removed by gel filtration (NAP™ 10 columns, Amersham Biosciences), and the sample was further concentrated with Biomax Ultrafree centrifugal filter device (Millipore, Bedford, MA). The reduced protein (40 μm) was reacted with either substrate peptide (50 μm) or buffer alone in a total volume of 100 μl in McIlvaine buffer at pH 7.0. The reaction was quenched with 50 mm N-ethylmaleimide or 1.1 m iodoacetamide (both from Sigma). The excess of N-ethylmaleimide/iodoacetamide was removed with pepClean™ C-18 spin columns (Pierce) according to the manufacturer's instructions. Proteins were eluted with 50%, acetonitrile and CH3COOH to a final concentration of 0.1% was added to samples. Molecular masses were measured with an electrospray ionization mass spectrometer (Micromass LCT, Manchester, UK) using positive ionization. Additional time-dependent trapping experiments were carried out using an RQF3 quenched-flow apparatus (KinTek, Austin, TX). The reduced protein (80 μm) was reacted with either substrate peptide (100 μm) or buffer alone in a total volume of 30 μl in McIlvaine buffer at pH 7.0. After the desired reaction time (0.01–1 s), the reaction was quenched with 0.5 m HCl, to ensure that the pH of the quenched product remained below pH 2.0 and was treated as previously.Stopped-flow Measurements—Stopped-flow experiments were performed with a SF2004 stopped-flow apparatus (Kin-Tek) with 20 μm E. coli C14S Grx1 and 0.14 mg/ml (357 μm) Ellman's reagent in McIlvaine buffer at the desired pH (4.5–7.5). The absorbance at 412 nm was measured for 0.2 s after mixing the two reagents, and the pseudo first-order rate constants were calculated using KinTek StopFlow v9.06 software.RESULTSDetermination of the Deglutathionylation Activity—To directly monitor the deglutathionylation activity of Grx, a glutathionylated substrate peptide was designed. The criteria for the design were that the peptide should contain a single glutathionylated cysteine residue with an adjacent fluorescent group. The peptide would also need to be small (for ease of synthesis) and water-soluble and contain no residues with charged side chains to eliminate any pH dependence of reaction rates from effects on the peptide. A similar strategy has previously been utilized to generate peptide substrates to monitor disulfide oxidation (34Ruddock L.W. Hirst T.R. Freedman R.B. Biochem. J. 1996; 315: 1001-1005Crossref PubMed Scopus (76) Google Scholar). The peptide synthesized here, SQL-WC(glutathione)LSN, had an emission maximum at 356 nm (Fig. 2A), consistent with an aqueous exposed tryptophan. Upon removal of the glutathione, i.e. the reduction of the cysteine moiety, a 38% increase in total fluorescence was observed with no shift in the emission maximum. This change is consistent with quenching of the fluorescence of tryptophan residues by adjacent disulfide bonds (35Neves-Petersen M.T. Grycynski Z. Lakowicz J. Fojan P. Pedersen S. Petersen E. Petersen S. Protein Sci. 2002; 11: 588-600Crossref PubMed Scopus (127) Google Scholar). Thus, deglutathionylation of the peptide could be measured directly in real time by monitoring the change in fluorescence at 356 nm in the presence of reducing agents such as GSH. At pH 7.0 in McIlvaine buffer, the non-catalyzed reaction was very slow, just 3% of the enzyme-catalyzed reaction with 20 nm E. coli Grx1 and 1 mm GSH (Fig. 2B). Since one of the products of the reaction, GSSG, may reglutathionylate the deglutathionylated peptide substrate, glutathione reductase and NADPH were added to the reaction to remove the GSSG formed.FIGURE 2Fluorescence analysis of peptide deglutathionylation. A, typical emission spectra for glutathionylated (– – –) and deglutathionylated (——) peptide (5 μm) at pH 7. 0. B, representative time-dependent fluorescence profiles during the deglutathionylation of the substrate peptide catalyzed by 20 nm E. coli Grx1 (black) and the non-catalyzed reaction (gray). Concentrations were as follows: McIlvaine buffer, pH 7.0, [GSH] = 1 mm, [substrate peptide] = 5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of the Reaction Kinetics—To analyze the kinetics as a function of enzyme, substrate, and GSH concentrations, the initial rates of reaction were determined from each time course and plotted against the concentration of the varying substance. As expected, E. coli Grx1 showed a linear dependence of the initial rate of reaction on the enzyme concentration (Fig. 3A). In addition, the initial rate of reaction increased with increasing substrate concentrations and fitted well to the Michaelis-Menten equation with Km = 7.8 μm and kcat = 4.4 s–1 ([GSH] = 1 mm; Fig. 3B). In contrast, although an increase in the initial rate with increasing GSH concentration was observed, with a plateau at the highest GSH concentrations measured, the plot could not be fitted to the Michaelis-Menten equation and instead showed a sigmoidal shape ([substrate peptide] = 5 μm; Fig. 3C).FIGURE 3Analysis of the reaction kinetics of glutaredoxin-catalyzed deglutathionylation. A, linear dependence of the initial rate of peptide deglutathionylation on Grx1 concentration (McIlvaine buffer, pH 7.0, [GSH] = 1 mm, [substrate peptide] = 5 μm). Initial rates are expressed as mean ± S.D. B, variation of the initial rate of reaction with substrate concentration during peptide deglutathionylation catalyzed by 20 nm Grx1 (McIlvaine buffer, pH 7.0, [GSH] = 1 mm). Initial rates are expressed as mean ± S.D. The line of best fit is to the Michaelis-Menten equation, and the inset shows a linear fit to a Lineweaver-Burk plot. C, variation of the initial rate of reaction with GSH concentration during peptide deglutathionylation catalyzed by 20 nm Grx1 (McIlvaine buffer, pH 7.0, [substrate peptide] = 5 μm). Initial rates are expressed as mean ± S.D. The inset shows a Lineweaver-Burk plot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of Grx1C14S Mutant-catalyzed Deglutathionylation Reaction—Often, a sigmoidal shape for the dependence of the enzyme-catalyzed rate on the substrate concentration implies cooperativity of substrate binding; however, this is unlikely for glutaredoxin since it is a small monomeric enzyme with a single glutathione binding site (31Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (129) Google Scholar, 36Nordstrand K. Åslund F. Holmgren A. Otting G. Berndt K.D. J. Mol. Biol. 1999; 286: 541-552Crossref PubMed Scopus (112) Google Scholar). Instead, the sigmoidal shape for the GSH dependence of E. coli Grx1 activity probably represents the net effect of the partitioning of the Grx-GSH mixed disulfide intermediate between the formation of reduced or oxidized Grx and the re-reduction of oxidized Grx1 by GSH (Fig. 1). To test this hypothesis, the C-terminal active site C14S mutant of E. coli Grx1 was made since this mutant cannot proceed to the oxidized state, and thus, there should be no partitioning. Under standard conditions ([GSH] = 1 mm, [substrate] = 5 μm), the initial rate of the C14S mutant-catalyzed reaction was only 23% of that catalyzed by the wild-type enzyme. Similar to wild type, the C14S mutant showed a linear dependence of the initial rate of reaction on the enzyme concentration (Fig. 4A). Furthermore, the data with increasing substrate concentrations fitted well to the Michaelis-Menten equation with Km = 44 μm and kcat = 4.0 s–1 ([GSH] = 1 mm; Fig. 4B), although no plateau was observed due to the narrow range of possible substrate concentrations that could be used. In contrast to wild type, the increase observed in the initial rate with increasing GSH concentrations could be fitted to the Michaelis-Menten equation, giving Km = 48 μm and kcat = 0.36 s–1 ([substrate peptide] = 5 μm; Fig. 4C). A lower mid-point for glutathione dependence for the mutant (here Km = 48 μm) when compared with the wild-type enzyme (here mid-point ∼0.5 mm) has previously been reported for human Grx and assigned to the effects of the partitioning reaction to the oxidized state for the wild-type enzyme (31Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (129) Google Scholar).FIGURE 4Analysis of the reaction kinetics of C14S glutaredoxin-catalyzed deglutathionylation. A, linear dependence of the initial rate of peptide deglutathionylation on C14S Grx1 concentration (McIlvaine buffer, pH 7.0, [GSH] = 1 mm, [substrate peptide] = 5 μm). Initial rates are expressed as mean ± S.D. B, variation of the initial rate of reaction with substrate concentration during peptide deglutathionylation catalyzed by 20 nm C14S Grx1 (McIlvaine buffer, pH 7.0, [GSH] = 1 mm). Initial rates are expressed as mean ± S.D. The line of best fit is to the Michaelis-Menten equation, and the inset shows a linear fit to a Lineweaver-Burk plot. C, variation of the initial rate of reaction with GSH concentration during peptide deglutathionylation catalyzed by 20 nm C14S Grx1 (McIlvaine buffer, pH 7.0, [substrate peptide] = 5μm). Initial rates are expressed as mean ± S.D. The line of best fit is to the Michaelis-Menten equation, and the inset shows a linear fit to a Lineweaver-Burk plot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Intermediates in the Deglutathionylation Reaction—It is presumed (31Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (129) Google Scholar, 37Bushweller J.H. Billeter M. Holmgren A. Wüthrich K. J. Mol. Biol. 1994; 235: 1585-1597Crossref PubMed Scopus (119) Google Scholar) that the major intermediate in the deglutathionylation reaction catalyzed by Grx is the Grx-GSH mixed disulfide. To try to confirm this, experiments for trapping the reaction intermediate in equilibrium as well as quenched-flow reactions were undertaken with Grx1 and substrate peptide in the absence of GSH. However, analysis of the reaction products indicated only the presence of oxidized Grx1 (mass = 10637 Da), indicating that the nucleophilic attack by the C-terminal active site Cys residue on the Grx-GSH mixed disulfide was significantly faster than the nucleophilic attack by Grx1 on the glutathionylated substrate (Fig. 1). Since the C14S Grx1 mutant cannot undergo the same partitioning reaction, similar equilibrium and quenched-flow reactions were undertaken with this mutant. The results indicated that no Grx-peptide mixed disulfide (mass = 11572 Da) was observed, with only the Grx-GSH (mass = 10928 Da) mixed disulfide being formed. Under the test conditions used ([Grx1] = 80 μm, [substrate] = 100 μm), the half-time for this reaction was around 100 ms.Effects of Oxidized Glutathione—Since Grx forms mixed disulfides with GSH and not with peptide and since the Km for the C14S for GSH (48 μm) and glutathionylated-substrate (44 μm) are similar, it is likely that oxidized glutathione (GSSG) will act as a competitive substrate (i.e. glutathionylated glutathione) for the deglutathionylation reaction catalyzed by Grx. However, since the product of the reaction using GSSG as a substrate is GSSG, the net effect of this is that GSSG would act like a competitive inhibitor of the peptide deglutathionylation reaction. To test this, the deglutathionylation reactions were performed in the presence of varying amounts of GSSG and in the absence of glutathione reductase. Using changes in the absorbance of NADPH and glutathione reductase-catalyzed reduction of GSSG, the amount of GSSG in the GSH stock was calculate" @default.
W1964119106 created "2016-06-24" @default.
W1964119106 creator A5000545274 @default.
W1964119106 creator A5008406047 @default.
W1964119106 creator A5016746605 @default.
W1964119106 creator A5041896881 @default.
W1964119106 creator A5089178800 @default.
W1964119106 date "2006-11-01" @default.
W1964119106 modified "2023-09-29" @default.
W1964119106 title "Insights into Deglutathionylation Reactions" @default.
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