The bis(2-hydroxyethyl) disulfide (HEDS) steady-state assay is the most commonly used assay to measure the activity and kinetic parameters of glutaredoxins. Despite its widespread use, the HEDS assay is often misinterpreted, and the exact enzyme mechanism has remained unclear. Here we used stopped-flow kinetic measurements to show that the active-site cysteinyl residue of mono- and dithiol class I glutaredoxins from Saccharomyces cerevisiae and Plasmodium falciparum reduces HEDS with a second-order rate constant around 10 5 M –1 s –1 , regardless of the absence or presence of reduced glutathione (GSH). Thus, HEDS is a direct non-glutathione disulfide substrate of glutaredoxins. Its turnover neither requires the formation of a ternary complex between HEDS, GSH, and the glutaredoxin, nor the frequently emphasized non-enzymatic parallel reaction between HEDS and GSH. Competition between the non-enzymatic reaction and the direct enzyme-dependent reduction of HEDS can occur at high GSH and very low glutaredoxin concentrations but requires GSH-to-glutaredoxin ratios ≥10 5 . The GSH-dependent reduction of the obtained mixed disulfide between 2-mercaptoethanol and the glutaredoxin completes the first ping-pong reaction with second-order rate constants of 10 4 –10 5 M –1 s –1 . The product of this ping-pong reaction, GSSEtOH, is then converted in a second ping-pong reaction with GSH, yielding 2-mercaptoethanol and glutathione disulfide. Simulations with alternative kinetic models suggest that the sequential patterns in the HEDS assay originate from variable GSSEtOH steady-state concentrations. In summary, we elucidated the enzyme mechanism for the HEDS assay and provide recommendations for conducting the assay and for data interpretation.
Lang et al. (Wed,) studied this question.