title: Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: rate constants by competitive kinetics.
abstract: Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. Likely to be critical for both functions is a rapid reaction with hydrogen peroxide, typically with second-order rate constants higher than 10(5) M(-1) s(-1). Until recently, however, the values reported for these rate constants have been in the range of 10(4)-10(5) M(-1) s(-1), including those for cytosolic thioredoxin peroxidases I (Tsa1) and II (Tsa2) from Saccharomyces cerevisiae. To resolve this apparent paradox, we developed a competitive kinetic approach with horseradish peroxidase to determine the second-order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and allowed for the determination of the second-order rate constant of the reaction of Tsa1 and Tsa2 with peroxynitrite (k approximately 10(5) M(-1) s(-1)) and hydrogen peroxide (k approximately 10(7) M(-1) s(-1)) at pH 7.4, 25 degrees C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In addition to providing a useful method for studying thiol protein kinetics, our studies add to recent reports challenging the popular belief that peroxiredoxins are poor enzymes toward hydrogen peroxide, as compared with heme and selenium proteins.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Renata
authors.initials: R
authors.lastname: Ogusucu
authors.forename: Daniel
authors.initials: D
authors.lastname: Rettori
authors.forename: Daniela Cristina
authors.initials: DC
authors.lastname: Munhoz
authors.forename: Luis Eduardo Soares
authors.initials: LE
authors.lastname: Netto
authors.forename: Ohara
authors.initials: O
authors.lastname: Augusto
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000737:chemistry*; D054464:Peroxiredoxins; D030421:Peroxynitrous Acid / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D030421:Peroxynitrous Acid; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010544:Peroxidases; C496253:Tsa1 protein, S cerevisiae; C496254:Tsa2 protein, S cerevisiae; D054464:Peroxiredoxins; D003545:Cysteine