TY - JOUR
T1 - Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine
AU - Chang, Tong Shin
AU - Jeong, Woojin
AU - Woo, Hyun Ae
AU - Sun, Mi Lee
AU - Park, Sunjoo
AU - Sue, Goo Rhee
PY - 2004/12/3
Y1 - 2004/12/3
N2 - Peroxiredoxins (Prxs) are a family of peroxidases that reduce hydroperoxides. The cysteine residue in the active site of certain eukaryotic Prx enzymes undergoes reversible oxidation to sulfinic acid (Cys-SO 2H) during catalysis, and sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their K m values (1.8 mM and 1.2 μM, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (∼7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys-SO2H of Prx in A549 human cells.
AB - Peroxiredoxins (Prxs) are a family of peroxidases that reduce hydroperoxides. The cysteine residue in the active site of certain eukaryotic Prx enzymes undergoes reversible oxidation to sulfinic acid (Cys-SO 2H) during catalysis, and sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their K m values (1.8 mM and 1.2 μM, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (∼7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys-SO2H of Prx in A549 human cells.
UR - http://www.scopus.com/inward/record.url?scp=10944237769&partnerID=8YFLogxK
U2 - 10.1074/jbc.M409482200
DO - 10.1074/jbc.M409482200
M3 - Article
C2 - 15448164
AN - SCOPUS:10944237769
SN - 0021-9258
VL - 279
SP - 50994
EP - 51001
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 49
ER -