Mechanistic and Kinetic Details of Catalysis of Thiol-Disulfide Exchange by Glutaredoxins and Potential Mechanisms of Regulation

Abstract

Glutaredoxins are small, heat-stable proteins that exhibit a characteristic thioredoxin fold and a CXXC=S activesite motif. A variety of glutathione (GSH)-dependent catalytic activities have been attributed to the glutaredoxins, including reduction of ribonucleotide reductase, arsenate, and dehydroascorbate; assembly of iron sulfur cluster complexes; and protein glutathionylation and deglutathionylation. Catalysis of reversible protein glutathionylation by glutaredoxins has been implicated in regulation of redox signal transduction and sulfhydryl homeostasis in numerous contexts in health and disease. This forum review is presented in two parts. Part I is focused primarily on the mechanism of the deglutathionylation reaction catalyzed by prototypical dithiol glutaredoxins, especially human Grx1 and Grx2. Grx-catalyzed protein deglutathionylation proceeds by a nucleophilic, double-displacement mechanism in which rate enhancement is attributed to special reactivity of the low pK a cysteine at its active site, and to increased nucleophilicity of the second substrate, GSH. Glutaredoxins (and Grx domains) have been identified in most organisms, and many exhibit deglutathionylation or other activities or both. Further characterization according to glutathionyl selectivity, physiological substrates, and intracellular roles may lead to subclassification of this family of enzymes. Part II presents potential mechanisms for in vivo regulation of Grx activity, providing avenues for future studies. Antioxid. Redox Signal. 11, 1059-1081.Part I: Glutaredoxins and Catalysis of Thiol-Disulfide Exchange G lutaredoxins are GSH-disulfide oxidoreductases reported to catalyze a variety of GSH-dependent thioldisulfide exchange reactions including protein glutathionylation and deglutathionylation, turnover of ribonucleotide reductase, and reduction of dehydroascorbate and arsenate; and some glutaredoxins are also implicated in FeS cluster homeostasis (reviewed in refs. 68, 80, 81). Among the reported catalytic activities of the glutaredoxins, protein deglutathionylation (reduction of protein-glutathione mixed disulfides, protein-SSG) has received much attention because of its regulatory roles in redox signal transduction and sulfhydryl homeostasis (reviewed in refs. 23, 80). Glutathionylation is an oxidative posttranslational modification that occurs on some protein cysteines under basal conditions [e.g., b-actin (137), mitochondrial complex II (19)]; for others, it is a transient modification that occurs during oxidative stresses such as ischemia=reperfusion [e.g., a-actin (18), GAPDH (26), mitochondrial complex I (56)]. For many proteins, glutathionylation affects function, and thus the reversible glutathionylation of specific proteins has been implicated in regulation of cellular homeostasis in health and disease (reviewed in refs. 23, 80). Grx is the primary intracellular deglutathionylating enzyme in mammalian cells (21,52), and manipulation of Grx levels has been shown to affect protein glutathionylation status and, subs...

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