Systematic re-evaluation of the bis(2-hydroxyethyl)disulfide (HEDS) assay reveals an alternative mechanism and activity of glutaredoxins.
نویسندگان
چکیده
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.
منابع مشابه
Systematic re-evaluation of the bis(2-hydroxyethyl)disulfide (HEDS) assay reveals an alternative mechanism and activity of glutaredoxins† †Electronic supplementary information (ESI) available: Alternative evaluation of the HEDS assay with ScGrx7 (Fig. S1), HEDS and GSSEtOH assay with PfGrx (Fig. S2), enzyme parameters for the GSSEtOH assay with ScGrx7 (Table S1) and calculations of estimated reaction velocities and substrate concentrations (Tables S2–4). See DOI: 10.1039/c5sc01051a Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.
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عنوان ژورنال:
- Chemical science
دوره 6 7 شماره
صفحات -
تاریخ انتشار 2015