Systematic re-evaluation of the bis(2-hydroxyethyl)disulfide (HEDS) assay reveals an alternative mechanism and activity of glutaredoxins
The reduction of bis(2-hydroxyethyl)disulfide (HEDS) by reduced glutathione (GSH) is the most commonly used assay to analyze the presence and properties of enzymatically active glutaredoxins (Grx), a family of central redox proteins in eukaryotes and glutathione-utilizing prokaryotes. Enzymatically...
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| Hauptverfasser: | , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
19 May 2015
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| In: |
Chemical science
Year: 2015, Jahrgang: 6, Heft: 7, Pages: 3788-3796 |
| ISSN: | 2041-6539 |
| DOI: | 10.1039/C5SC01051A |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1039/C5SC01051A Verlag, lizenzpflichtig, Volltext: https://pubs.rsc.org/en/content/articlelanding/2015/sc/c5sc01051a 
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| Verfasserangaben: | Patricia Begas, Verena Staudacher and Marcel Deponte |
| Zusammenfassung: | The reduction of bis(2-hydroxyethyl)disulfide (HEDS) by reduced glutathione (GSH) is the most commonly used assay to analyze the presence and properties of enzymatically active glutaredoxins (Grx), a family of central redox proteins in eukaryotes and glutathione-utilizing prokaryotes. Enzymatically active Grx usually prefer glutathionylated disulfide substrates. These are converted via a ping-pong mechanism. Sequential kinetic patterns for the HEDS assay have therefore been puzzling since 1991. Here we established a novel assay and used the model enzyme ScGrx7 from yeast and PfGrx from Plasmodium falciparum to test several possible causes for the sequential kinetics such as pre-enzymatic GSH depletion, simultaneous binding of a glutathionylated substrate and GSH, as well as substrate or product inhibition. Furthermore, we analyzed the non-enzymatic reaction between HEDS and GSH by HPLC and mass spectrometry suggesting that such a reaction is too slow to explain high Grx activities in the assay. The most plausible interpretation of our results is a direct Grx-catalyzed reduction of HEDS. Physiological implications of this alternative mechanism and of the Grx-catalyzed reduction of non-glutathione disulfide substrates are discussed. |
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| Beschreibung: | Gesehen am 04.08.2020 |
| Beschreibung: | Online Resource |
| ISSN: | 2041-6539 |
| DOI: | 10.1039/C5SC01051A |