title: Kinetic and mechanistic characterization and versatile catalytic properties of mammalian glutaredoxin 2: implications for intracellular roles.
abstract: Glutaredoxin (Grx)-catalyzed deglutathionylation of protein-glutathione mixed disulfides (protein-SSG) serves important roles in redox homeostasis and signal transduction, regulating diverse physiological and pathophysiological events. Mammalian cells have two Grx isoforms: Grx1, localized to the cytosol and mitochondrial intermembrane space, and Grx2, localized primarily to the mitochondrial matrix [Pai, H. V., et al. (2007) Antioxid. Redox Signaling 9, 2027-2033]. The catalytic behavior of Grx1 has been characterized extensively, whereas Grx2 catalysis is less well understood. We observed that human Grx1 and Grx2 exhibit key catalytic similarities, including selectivity for protein-SSG substrates and a nucleophilic, double-displacement, monothiol mechanism exhibiting a strong commitment to catalysis. A key distinction between Grx1- and Grx2-mediated deglutathionylation is decreased catalytic efficiency ( k cat/ K M) of Grx2 for protein deglutathionylation (due primarily to a decreased k cat), reflecting a higher p K a of its catalytic cysteine, as well as a decreased enhancement of nucleophilicity of the second substrate, GSH. As documented previously for hGrx1 [Starke, D. W., et al. (2003) J. Biol. Chem. 278, 14607-14613], hGrx2 catalyzes glutathione-thiyl radical (GS (*)) scavenging, and it also mediates GS transfer (protein S-glutathionylation) reactions, where GS (*) serves as a superior glutathionyl donor substrate for formation of GAPDH-SSG, compared to GSNO and GSSG. In contrast to its lower k cat for deglutathionylation reactions, Grx2 promotes GS-transfer to the model protein substrate GAPDH at rates equivalent to those of Grx1. Estimation of Grx1 and Grx2 concentrations within mitochondria predicts comparable deglutathionylation activities within the mitochondrial subcompartments, suggesting localized regulatory functions for both isozymes.
authors.affiliation: Department of Pharmacology, Case Western Reserve University, School of Medicine, 2109 Adelbert Road, Cleveland, Ohio 44106-4965, USA.
authors.forename: Molly M
authors.initials: MM
authors.lastname: Gallogly
authors.forename: David W
authors.initials: DW
authors.lastname: Starke
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Leonberg
authors.forename: Susan M English
authors.initials: SM
authors.lastname: Ospina
authors.forename: John J
authors.initials: JJ
authors.lastname: Mieyal
mesh_terms: D000818:Animals; D002417:Cattle; D054477:Glutaredoxins / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: C516005:GLRX protein, human; C516008:GLRX2 protein, human; C516009:Glrx2 protein, mouse; D054477:Glutaredoxins; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D019803:Glutathione Disulfide