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• W2600965896 abstract "Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is large amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 105 and 2.3 × 106 m−1 s−1 for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme; then it oxidizes the peroxidatic cysteine, and finally, the rate-limiting disulfide bond is formed. Of relevance, the disulfide bond formation was much slower in Prx2 (k3 = 0.31 s−1) than Prx1 (k3 = 14.9 s−1). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid. Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is large amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 105 and 2.3 × 106 m−1 s−1 for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme; then it oxidizes the peroxidatic cysteine, and finally, the rate-limiting disulfide bond is formed. Of relevance, the disulfide bond formation was much slower in Prx2 (k3 = 0.31 s−1) than Prx1 (k3 = 14.9 s−1). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid. Uric acid is the end product of purine metabolism in humans. Urate, the anionic form of uric acid (pKa 5.4), accumulates in plasma in concentrations ranging from 50 to 420 μm in healthy individuals. The gene silencing of the enzyme uricase has been suggested to be an evolutionary advantage because urate is a facile electron donor (one-electron reduction potential = 0.59 V, pH 7.0, HU-·, H+/UH2−) and therefore a powerful antioxidant (1Simic M.G. Jovanovic S.V. Antioxidation mechanisms of uric acid.J. Am. Chem. Soc. 1989; 111: 5778-5782Crossref Scopus (239) Google Scholar, 2Oda M. Satta Y. Takenaka O. Takahata N. Loss of urate oxidase activity in hominoids and its evolutionary implications.Mol. Biol. Evol. 2002; 19: 640-653Crossref PubMed Scopus (296) Google Scholar). 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The NLRP3 inflammasome: a sensor for metabolic danger?.Science. 2010; 327: 296-300Crossref PubMed Scopus (853) Google Scholar). However, soluble urate also exerts pro-inflammatory effects by stimulating the oxidative burst in adipocytes (11Sautin Y.Y. Nakagawa T. Zharikov S. Johnson R.J. Adverse effects of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative stress.Am. J. Physiol. Cell Physiol. 2007; 293: C584-C596Crossref PubMed Scopus (557) Google Scholar, 18Baldwin W. McRae S. Marek G. Wymer D. Pannu V. Baylis C. Johnson R.J. Sautin Y.Y. Hyperuricemia as a mediator of the proinflammatory endocrine imbalance in the adipose tissue in a murine model of the metabolic syndrome.Diabetes. 2011; 60: 1258-1269Crossref PubMed Scopus (313) Google Scholar). Urate can be oxidized by the pro-inflammatory enzymes myeloperoxidase (MPO) 3The abbreviations used are: MPOmyeloperoxidaseLPOlactoperoxidaseANOVAanalysis of variancePrxperoxiredoxinDTNB5,5′-dithiobis(nitrobenzoic acid)DTPAdiethylenetriaminepentaacetic acidNEMN-ethylmaleimideMPmobile phase. and lactoperoxidase (LPO) to generate urate free radical and urate hydroperoxide (19Meotti F.C. Jameson G.N. Turner R. Harwood D.T. Stockwell S. Rees M.D. Thomas S.R. Kettle A.J. Urate as a physiological substrate for myeloperoxidase: implications for hyperuricemia and inflammation.J. Biol. Chem. 2011; 286: 12901-12911Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 20Seidel A. Parker H. Turner R. Dickerhof N. Khalilova I.S. Wilbanks S.M. Kettle A.J. Jameson G.N. Uric acid and thiocyanate as competing substrates of lactoperoxidase.J. Biol. Chem. 2014; 289: 21937-21949Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Considering the levels of urate in human biological fluids, as well as the concentration of MPO and LPO in inflammatory environments (21Ihalin R. Loimaranta V. Tenovuo J. Origin, structure, and biological activities of peroxidases in human saliva.Arch. Biochem. Biophys. 2006; 445: 261-268Crossref PubMed Scopus (156) Google Scholar, 22Klebanoff S.J. Peroxidases in Chemistry and Biology.in: Everse J. Everse K.E. Grisham M.B. Myeloperoxidase: Occurrence and Biological Function. CRC Press, Boca Raton, FL1991: 1-35Google Scholar), a significant amount of urate hydroperoxide might be formed during inflammatory oxidative burst. Urate hydroperoxide oxidizes methionine and cysteine and reacts with glutathione at a rate constant of 13.8 m−1 s−1 (23Patrício E.S. Prado F.M. da Silva R.P. Carvalho L.A. Prates M.V. Dadamos T. Bertotti M. Di Mascio P. Kettle A.J. Meotti F.C. Chemical characterization of urate hydroperoxide, a pro-oxidant intermediate generated by urate oxidation in inflammatory and photoinduced processes.Chem. Res. Toxicol. 2015; 28: 1556-1566Crossref PubMed Scopus (19) Google Scholar). Therefore, proteins that contain thiol groups are putative urate hydroperoxide targets. myeloperoxidase lactoperoxidase analysis of variance peroxiredoxin 5,5′-dithiobis(nitrobenzoic acid) diethylenetriaminepentaacetic acid N-ethylmaleimide mobile phase. Peroxiredoxins are ubiquitous cysteine-dependent peroxidases. They reduce hydrogen peroxide, peroxynitrite, and organic peroxides at extremely high rates (24Peskin A.V. Low F.M. Paton L.N. Maghzal G.J. Hampton M.B. Winterbourn C.C. The high reactivity of peroxiredoxin 2 with H(2)O(2) is not reflected in its reaction with other oxidants and thiol reagents.J. Biol. Chem. 2007; 282: 11885-11892Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 25Cox A.G. Peskin A.V. Paton L.N. 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Kinetic studies on peroxynitrite reduction by peroxiredoxins.Methods Enzymol. 2008; 441: 173-196Crossref PubMed Scopus (61) Google Scholar, 30Ogusucu R. Rettori D. Munhoz D.C. Netto L.E. Augusto O. Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: rate constants by competitive kinetics.Free Radic. Biol. Med. 2007; 42: 326-334Crossref PubMed Scopus (158) Google Scholar, 31Tairum C.A. Santos M.C. Breyer C.A. Geyer R.R. Nieves C.J. Portillo-Ledesma S. Ferrer-Sueta G. Toledo Jr, J.C. Toyama M.H. Augusto O. Netto L.E. de Oliveira M.A. Catalytic Thr or Ser residue modulates structural switches in 2-Cys peroxiredoxin by distinct mechanisms.Sci. Rep. 2016; 633133Crossref PubMed Scopus (34) Google Scholar). There are six human Prxs (Prx1 to Prx6) that vary in their intracellular location and catalytic mechanisms. Prx1 and Prx2 are typical 2-Cys proteins present in cytosol (32Rhee S.G. Woo H.A. Kil I.S. Bae S.H. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides.J. Biol. Chem. 2012; 287: 4403-4410Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). Their enzymatic cycle involves oxidation of the peroxidatic cysteine (CP) to a sulfenic acid, followed by disulfide bond formation with the resolving cysteine (CR) of another subunit, resulting in a head-to-tail covalent dimer. Their basic functional unit is a homodimer during the entire catalytic cycle, and they can assemble into high molecular weight species in cells (31Tairum C.A. Santos M.C. Breyer C.A. Geyer R.R. Nieves C.J. Portillo-Ledesma S. Ferrer-Sueta G. Toledo Jr, J.C. Toyama M.H. Augusto O. Netto L.E. de Oliveira M.A. Catalytic Thr or Ser residue modulates structural switches in 2-Cys peroxiredoxin by distinct mechanisms.Sci. Rep. 2016; 633133Crossref PubMed Scopus (34) Google Scholar, 33Wood Z.A. Poole L.B. Hantgan R.R. Karplus P.A. Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins.Biochemistry. 2002; 41: 5493-5504Crossref PubMed Scopus (299) Google Scholar). During the catalytic cycle, the disulfide bond formation can be overcome if the oxidant concentration is sufficiently high to compete for the sulfenic acid to yield the sulfinic and sulfonic acid-hyperoxidized Prx (33Wood Z.A. Poole L.B. Hantgan R.R. Karplus P.A. Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins.Biochemistry. 2002; 41: 5493-5504Crossref PubMed Scopus (299) Google Scholar, 34Hall A. Karplus P.A. Poole L.B. Typical 2-Cys peroxiredoxins–structures, mechanisms and functions.FEBS J. 2009; 276: 2469-2477Crossref PubMed Scopus (363) Google Scholar, 35Peskin A.V. Dickerhof N. Poynton R.A. Paton L.N. Pace P.E. Hampton M.B. Winterbourn C.C. Hyperoxidation of peroxiredoxins 2 and 3: rate constants for the reactions of the sulfenic acid of the peroxidatic cysteine.J. Biol. Chem. 2013; 288: 14170-14177Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 36Karplus P.A. A primer on peroxiredoxin biochemistry.Free Radic. Biol. Med. 2015; 80: 183-190Crossref PubMed Scopus (86) Google Scholar). Hyperoxidation of Prx significantly limits the turnover of the enzyme. The intermolecular disulfide bond is mainly reduced by the thioredoxin-thioredoxin reductase system (32Rhee S.G. Woo H.A. Kil I.S. Bae S.H. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides.J. Biol. Chem. 2012; 287: 4403-4410Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar) and in some cases by glutaredoxin (37Hanschmann E.M. Lönn M.E. Schütte L.D. Funke M. Godoy J.R. Eitner S. Hudemann C. Lillig C.H. Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3.J. Biol. Chem. 2010; 285: 40699-40705Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 38Peskin A.V. Pace P.E. Behring J.B. Paton L.N. Soethoudt M. Bachschmid M.M. Winterbourn C.C. Glutathionylation of the active site cysteines of peroxiredoxin 2 and recycling by glutaredoxin.J. Biol. Chem. 2016; 291: 3053-3062Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In contrast, the hyperoxidized form can only be reduced by sulfiredoxin, a slower process that consumes ATP (39Woo H.A. Jeong W. Chang T.S. Park K.J. Park S.J. Yang J.S. Rhee S.G. Reduction of cysteine sulfinic acid by sulfiredoxin is specific to 2-cys peroxiredoxins.J. Biol. Chem. 2005; 280: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 40Jeong W. Park S.J. Chang T.S. Lee D.Y. Rhee S.G. Molecular mechanism of the reduction of cysteine sulfinic acid of peroxiredoxin to cysteine by mammalian sulfiredoxin.J. Biol. Chem. 2006; 281: 14400-14407Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Prx1 and Prx2 share 91% of homology and 78% of identity in their amino acid sequences (41Lee W. Choi K.S. Riddell J. Ip C. Ghosh D. Park J.H. Park Y.M. Human peroxiredoxin 1 and 2 are not duplicate proteins: the unique presence of CYS83 in Prx1 underscores the structural and functional differences between Prx1 and Prx2.J. Biol. Chem. 2007; 282: 22011-22022Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Despite the similarities, Prx1 has four cysteine residues (Cys-52, Cys-71, Cys-83, and Cys-173) and Prx2 has three (Cys-51, Cys-70, and Cys-172). Indeed, these enzymes are not redundant, and structural differences provide unique functions (42Neumann C.A. Krause D.S. Carman C.V. Das S. Dubey D.P. Abraham J.L. Bronson R.T. Fujiwara Y. Orkin S.H. Van Etten R.A. Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression.Nature. 2003; 424: 561-565Crossref PubMed Scopus (636) Google Scholar, 43Lee T.H. Kim S.U. Yu S.L. Kim S.H. Park D.S. Moon H.B. Dho S.H. Kwon K.S. Kwon H.J. Han Y.H. Jeong S. Kang S.W. Shin H.S. Lee K.K. Rhee S.G. Yu D.Y. Peroxiredoxin II is essential for sustaining life span of erythrocytes in mice.Blood. 2003; 101: 5033-5038Crossref PubMed Scopus (331) Google Scholar). Prx1 and Prx2 are oxidized at extraordinary high rates by hydrogen peroxide and can transfer these oxidizing equivalents to another signaling protein through thiol-disulfide exchange reactions, which seems to be relevant in redox signaling (44Wood Z.A. Poole L.B. Karplus P.A. Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling.Science. 2003; 300: 650-653Crossref PubMed Scopus (1142) Google Scholar, 45Winterbourn C.C. Hampton M.B. Redox biology: signaling via a peroxiredoxin sensor.Nat. Chem. Biol. 2015; 11: 5-6Crossref PubMed Scopus (65) Google Scholar, 46Sobotta M.C. Liou W. Stöcker S. Talwar D. Oehler M. Ruppert T. Scharf A.N. Dick T.P. Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling.Nat. Chem. Biol. 2015; 11: 64-70Crossref PubMed Scopus (425) Google Scholar, 47Jarvis R.M. Hughes S.M. Ledgerwood E.C. Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells.Free Radic. Biol. Med. 2012; 53: 1522-1530Crossref PubMed Scopus (201) Google Scholar). For instance, Prx2 forms a redox relay with STAT3 (signal transducer and activator of transcription 3), inhibiting STAT3 migration to the nucleus (46Sobotta M.C. Liou W. Stöcker S. Talwar D. Oehler M. Ruppert T. Scharf A.N. Dick T.P. Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling.Nat. Chem. Biol. 2015; 11: 64-70Crossref PubMed Scopus (425) Google Scholar). Prx1, however, can transfer their oxidizing equivalents to the ASK1 (apoptosis-regulating kinase-1 signaling), resulting in the phosphorylation of p38 and activation of apoptosis (47Jarvis R.M. Hughes S.M. Ledgerwood E.C. Peroxiredoxin 1 functions as a signal peroxidase to receive, transduce, and transmit peroxide signals in mammalian cells.Free Radic. Biol. Med. 2012; 53: 1522-1530Crossref PubMed Scopus (201) Google Scholar). In this study, we investigated the kinetics and mechanism of the oxidation of Prx1 and Prx2 by urate hydroperoxide. The observed high rate constants indicate that these proteins might be preferential targets of urate hydroperoxide in cells. Hyperoxidation of Prxs only occurred at high concentrations of urate hydroperoxide and may not be relevant in vivo. Urate hydroperoxide oxidized Prx2 in intact erythrocytes to the same extent as hydrogen peroxide. In conclusion, this study contributes to the understanding of the catalytic mechanism of 2-Cys Prxs in the reduction of an organic peroxide formed during inflammatory processes. Initially, we sought to identify whether Prx1 and Prx2 would be oxidized by urate hydroperoxide. The enzymes were incubated with different concentrations of urate hydroperoxide, and the oxidation of Prxs was evaluated by the appearance of disulfide bond dimers (∼42 kDa) in non-reducing SDS-PAGE. An equimolar concentration of urate hydroperoxide and Prx was enough to convert almost all monomers into dimers (Fig. 1). Next, we investigated the rate of Prx oxidation by urate hydroperoxide taking advantage of fluorescence changes during Prx oxidation (28Trujillo M. Clippe A. Manta B. Ferrer-Sueta G. Smeets A. Declercq J.P. Knoops B. Radi R. Pre-steady state kinetic characterization of human peroxiredoxin 5: taking advantage of Trp84 fluorescence increase upon oxidation.Arch. Biochem. Biophys. 2007; 467: 95-106Crossref PubMed Scopus (136) Google Scholar, 48Winterbourn C.C. Peskin A.V. Kinetic approaches to measuring peroxiredoxin reactivity.Mol. Cells. 2016; 39: 26-30Crossref PubMed Scopus (34) Google Scholar, 49Parsonage D. Nelson K.J. Ferrer-Sueta G. Alley S. Karplus P.A. Furdui C.M. Poole L.B. Dissecting peroxiredoxin catalysis: separating binding, peroxidation, and resolution for a bacterial AhpC.Biochemistry. 2015; 54: 1567-1575Crossref PubMed Scopus (47) Google Scholar). As described previously for typical 2-Cys Prx (31Tairum C.A. Santos M.C. Breyer C.A. Geyer R.R. Nieves C.J. Portillo-Ledesma S. Ferrer-Sueta G. Toledo Jr, J.C. Toyama M.H. Augusto O. Netto L.E. de Oliveira M.A. Catalytic Thr or Ser residue modulates structural switches in 2-Cys peroxiredoxin by distinct mechanisms.Sci. Rep. 2016; 633133Crossref PubMed Scopus (34) Google Scholar, 48Winterbourn C.C. Peskin A.V. Kinetic approaches to measuring peroxiredoxin reactivity.Mol. Cells. 2016; 39: 26-30Crossref PubMed Scopus (34) Google Scholar, 49Parsonage D. Nelson K.J. Ferrer-Sueta G. Alley S. Karplus P.A. Furdui C.M. Poole L.B. Dissecting peroxiredoxin catalysis: separating binding, peroxidation, and resolution for a bacterial AhpC.Biochemistry. 2015; 54: 1567-1575Crossref PubMed Scopus (47) Google Scholar), a two-phase fluorescent profile was observed, displaying a rapid decrease and a subsequent slower increase in fluorescence intensity (Fig. 2A). Initially, the first rapid decay was fitted by a single exponential equation (Figs. 2B and 3A). The observed rate constants for the first rapid phase were linearly dependent on urate hydroperoxide concentration, and the determined second-order rate constants were k1 = 2.26 ± 0.13 × 106 m−1 s−1 for Prx2 (Fig. 2C) and k1 = 4.90 ± 0.47 × 105 m−1 s−1 for Prx1 (Fig. 3B).Figure 3Kinetics of the oxidation of WTPrx1 and Prx1C83S/C173S by urate hydroperoxide. A, first rapid phase of the reaction of pre-reduced Prx1 (5 μm) incubated with 35 μm urate hydroperoxide in 50 mm sodium phosphate buffer (pH 7.4; 22 °C). Reactions were monitored over time by the variation of intrinsic protein fluorescence (λex = 280 nm, emission filter >330 nm) in the stopped-flow instrument. Observed rate constants (kobs) were calculated by single exponential equation. B, plot of kobs of the first rapid phase reaction of Prx1 versus urate hydroperoxide concentration. The second-order rate constant was calculated from this slope. C, fluorescence increase during the slow phase of the reaction of Prx1 (5 μm) with 240 μm urate hydroperoxide. Observed rate constants (kobs) were calculated by single exponential equation. D, plot of kobs of the slow phase of the reaction of Prx1 versus urate hydroperoxide. Non-linear curve was best fitted with a hyperbolic equation. E, first rapid phase of the reaction of pre-reduced Prx1 that was mutated at the resolving (Cys-173) and non-catalytic cysteine (Cys-83) (Prx1C83S/C173S, 5 μm) incubated with 65 μm urate hydroperoxide in 50 mm sodium phosphate buffer (pH 7.4; 22 °C). Observed rate constants (kobs) were calculated by single exponential plus straight line equation. F, plot of kobs of the first rapid phase of the reaction of Prx1 versus urate hydroperoxide concentration. V, voltage.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The linear fitting from kobs versus urate hydroperoxide concentration showed a clear non-zero y-intercept for Prx2, indicating that the first phase is reversible. The value of the y-intercept (k−1) was 99.0 ± 3.0 s−1 (Fig. 2C). The first fast decrease in Prx2 fluorescence (Fig. 2B) was followed by a slow linear decrease (Fig. 2B, inset). The rate of this linear decrease was independent of urate hydroperoxide concentration and could be the actual oxidation of the peroxidatic thiol to sulfenic acid. In contrast, this slow linear decrease in fluorescence was not observed for Prx1. In addition, when the kobs of the first rapid fluorescence decrease of Prx1 was plotted against urate hydroperoxide concentrations, the linear fitting presented a close to zero y-intercept, 1.47 ± 2.12 s−1 (Fig. 3B). Unlike the first phase, the kobs of the second phase levels off with increasing concentrations of urate hydroperoxide (Figs. 2E and 3D). The plot of kobs versus substrate concentration was well fitted to a non-linear hyperbolic equation. The limiting rate constants (k3) were 0.31 ± 0.01 s−1 for Prx2 (Fig. 2, D and E) and 14.9 ± 1.01 s−1 for Prx1 (Fig. 3, C and D). No changes in Prx fluorescence were observed in the absence of oxidants or when thiols from Prxs were alkylated with N-ethylmaleimide (NEM) (supplemental Fig. S1). Kinetics of His-tagged Prx2 or His-tagged free Prx2 were very similar: 2.26 ± 0.13 × 106 and 1.80 ± 0.12 × 106 m−1s−1 for the first phase; the y-intercept (k−1) was 99.0 ± 3.0 and 43.0 ± 1.2 s−1 and k3 = 0.31 ± 0.01 and 0.38 ± 0.03 s−1, respectively (data not shown). According to a three-step model reported for the oxidation of AhpC by hydrogen peroxide (43Lee T.H. Kim S.U. Yu S.L. Kim S.H. Park D.S. Moon H.B. Dho S.H. Kwon K.S. Kwon H.J. Han Y.H. Jeong S. Kang S.W. Shin H.S. Lee K.K. Rhee S.G. Yu D.Y. Peroxiredoxin II is essential for sustaining life span of erythrocytes in mice.Blood. 2003; 101: 5033-5038Crossref PubMed Scopus (331) Google Scholar), the first rapid decay of fluorescence would correspond to the binding of the enzyme to the peroxide substrate and the oxidation of CP. Thus, k1 represents the enzyme-substrate complex formation and k−1 the complex dissociation. The slow fluorescence increase would represent the disulfide bond formation. To confirm that the fluorescence increase was due to the disulfide formation also in human Prxs, we used a double mutant Prx1 where the resolving (Cys-173) and the non-catalytic (Cys-83) cysteine were replaced by serine (Prx1C83S/C173S). As expected, upon oxidation by urate hydroperoxide, the intrinsic fluorescence of this mutant exhibited a rapid decay but lacked the second slow phase (Fig. 3E), suggesting that the rise in fluorescence observed in WTPrx1 was due to the disulfide bond formation. The fluorescence decay for Prx1C83S/C173S presented a similar feature as for WTPrx1, and the kobs increased linearly with increasing concentrations of urate hydroperoxide (Fig. 3E). However, the second-order rate constant (k1) for this reaction was 1 order of magnitude lower for Prx1C83S/C173S (4.50 ± 0.18 × 104 m−1 s−1, Fig. 3F) than for WTPrx1 and the k−1 was 0.44 ± 0.04 s−1. The second-order rate constant of the reaction of Prx2 with urate hydroperoxide was only 1–2 orders of magnitude lower than that with hydrogen peroxide 0.2–1.3 × 108 m−1 s−1 (25Cox A.G. Peskin A.V. Paton L.N. Winterbourn C.C. Hampton M.B. Redox potential and peroxide reactivity of human peroxiredoxin 3.Biochemistry. 2009; 48: 6495-6501Crossref PubMed Scopus (100) Google Scholar, 26Manta B. Hugo M. Ortiz C. Ferrer-Sueta G. Trujillo M. Denicola A. The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2.Arch. Biochem. Biophys. 2009; 484: 146-154Crossref PubMed Scopus (154) Google Scholar), showing that Prx2 might be a physiological target to urate hydroperoxide. There are no rate constants reported for the reaction of Prx1 and hydrogen peroxide. Therefore, we carried out fluorescence experiments to determine it. The reaction of Prx1 with hydrogen peroxide was faster than with urate hydroperoxide, so we had to use sub-stoichiometric concentrations of substrate and employ an initial rate approach. By plotting the initial rates versus hydrogen peroxide concentration, the second-order rate constant was 3.80 ± 0.15 × 107 m−1 s−1 (Fig. 4A). The linear plot intercepted the y axis at 4.42 ± 1.85 μm−1 s−1. The increase in fluorescence after mixing Prx1 with hydrogen peroxide followed first-order kinetics and was independent of hydrogen peroxide concentration. The kobs of this step was 9.0 ± 0.2 s−1 (Fig. 4B), which is very similar to that calculated when the substrate was urate hydroperoxide (14.9 ± 1.01 s−1, Fig. 3D). The independency of the nature of peroxide in this rate constant is additional evidence that the return in fluorescence is due to a disulfide bond formation with the resolving cysteine rather than hyperoxidation. We" @default.
• W2600965896 created "2017-04-07" @default.
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• W2600965896 date "2017-05-01" @default.
• W2600965896 modified "2023-10-16" @default.
• W2600965896 title "Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2" @default.
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