Abstract
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-SO2H) 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 Km values (1.8 mM and 1.2 microM, 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 (approximately 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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Adenosine Triphosphate / chemistry
-
Amino Acid Sequence
-
Animals
-
Binding Sites
-
Cell Line, Tumor
-
Cloning, Molecular
-
Cysteine / analogs & derivatives*
-
Cysteine / chemistry*
-
DNA, Complementary / metabolism
-
Dose-Response Relationship, Drug
-
Escherichia coli / metabolism
-
Glutathione / chemistry
-
Glutathione / metabolism
-
Glutathione Transferase / metabolism
-
Guanosine Triphosphate / chemistry
-
HeLa Cells
-
Humans
-
Hydrogen-Ion Concentration
-
Kinetics
-
Mice
-
Models, Chemical
-
Molecular Sequence Data
-
Oxidation-Reduction
-
Oxidoreductases / chemistry*
-
Oxidoreductases Acting on Sulfur Group Donors / chemistry*
-
Oxygen / chemistry
-
Peroxidases / chemistry*
-
Peroxidases / physiology*
-
Peroxiredoxins
-
RNA Interference
-
Rats
-
Recombinant Proteins / chemistry
-
Saccharomyces cerevisiae / metabolism
-
Saccharomyces cerevisiae Proteins
-
Schizosaccharomyces / metabolism
-
Sequence Homology, Amino Acid
-
Temperature
-
Thioredoxins / chemistry
-
Time Factors
-
Tissue Distribution
Substances
-
DNA, Complementary
-
Recombinant Proteins
-
Saccharomyces cerevisiae Proteins
-
Thioredoxins
-
Guanosine Triphosphate
-
Adenosine Triphosphate
-
Oxidoreductases
-
Peroxidases
-
Peroxiredoxins
-
Oxidoreductases Acting on Sulfur Group Donors
-
SRX1 protein, S cerevisiae
-
sulfiredoxin protein, mouse
-
Glutathione Transferase
-
Glutathione
-
Cysteine
-
Oxygen
-
cysteine sulfinic acid