NDLI: Evidence for the formation of a covalent thiosulfinate intermediate with peroxiredoxin in the catalytic mechanism of sulfiredoxin.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Béchade, Guillaume Kriznik, Alexandre Rahuel-clermont, Sophie Roussel, Xavier Branlant, Guy Sanglier-cianferani, Sarah Van Dorsselaer, Alain
Description	Author Affiliation: Roussel X ( Unité Mixte de Recherche, CNRS-UHP 7567, Maturation des Acide Ribonucléique et Enzymologie Moléculaire, Nancy Université, Faculté des Sciences, Bld. des Aiguillettes, BP 239, Vandoeuvre-lès-Nancy 54506, France.)
Abstract	The typical 2-Cys peroxiredoxins are thiol-peroxidases involved in the physiology of hydrogen peroxide not only as a toxic but also as a signaling molecule. Coordination of these functions depends on the sulfinylation of the catalytic Cys, a modification reversed by ATP-dependent sulfiredoxin, which specifically reduces the sulfinic acid group of overoxidized 2-Cys peroxiredoxins into a sulfenic acid. Sulfiredoxin was originally proposed to operate by covalent catalysis, with formation of a peroxiredoxin-sulfiredoxin intermediate linked by a thiosulfinate bond between the catalytic Cys of both partners, a hypothesis rejected by a study of the human enzyme. To settle the argument, we investigated the catalytic mechanism of Saccharomyces cerevisiae sulfiredoxin, by the characterization of the nature and kinetics of formation of the protein species formed between sulfiredoxin and its substrate in the presence of ATP, using mutants of the non-essential Cys residues of both proteins. We observed the formation of a dithiothreitol-reducible peroxiredoxin-sulfiredoxin species using SDS-PAGE and Western blot analysis, and its mass was shown to correspond to a thiosulfinate complex by high resolution mass spectrometry coupled to liquid chromatography. We next measured indirectly and directly a rate constant of formation of the thiosulfinate species of approximately 2 min(-1), for both wild-type and mutant sulfiredoxins, at least equal to the steady-state rate constant of the reaction, with a stoichiometry of 1:1 relative to peroxiredoxin. Taken altogether, our results strongly argue in favor of the formation of a covalent thiosulfinate peroxiredoxin-sulfiredoxin species as an intermediate on the catalytic pathway.
ISSN	00219258
e-ISSN	1083351X
Journal	Journal of Biological Chemistry
Issue Number	33
Volume Number	283
Language	English
Publisher	American Society for Biochemistry and Molecular Biology (United States)
Publisher Date	2008-08-15
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Oxidoreductases Acting On Sulfur Group Donors Metabolism Peroxiredoxins Sulfinic Acids Adenosine Diphosphate Adenosine Triphosphate Animals Catalysis Cysteine Escherichia Coli Escherichia Coli Proteins Horses Kinetics Myoglobin Chemistry Reactive Oxygen Species Thioredoxin-Disulfide Reductase Thioredoxins Research Support, Non-U.S. Gov't Biochemistry Molecular Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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