Dynamics of the glutathione/glutaredoxin system.

Abstract

The glutathione/glutaredoxin system is made up of glutaredoxins, glutathione (GSH) and glutathione reductase (GLR). Glutaredoxins, which are involved in essential cellular functions such as DNA synthesis, iron metabolism and iron-sulfur cluster assembly, become oxidised during their catalytic cycle and are reduced by GSH and GLR. Glutaredoxins also play a critical role in regulating the glutathionylation/deglutathionylation cycle. Under oxidative stress conditions, protein thiols may be glutathionylated and glutaredoxin activity is important for restoring the functions of these proteins. While the individual components of this system have been studied extensively, the dynamics of the system as a whole has not been described despite its importance in the glutathionylation/deglutathionylation process. Computational systems biology approaches could be used to describe this type of regulation but the kinetic mechanism used by glutaredoxins for deglutathionylation is unclear as a monothiol and a dithiol mechanism have both been proposed for glutaredoxin activity. The in vitro data supporting these mechanisms have been contradictory with a number of discrepancies observed in the literature, including contrasting activities of mutant glutaredoxin Cxx(C→S) and wild-type glutaredoxins. Further, Lineweaver-Burk plots showed a curved line pattern in some studies, while other studies reported a linear pattern in response to GSH. Finally, analyses of the Lineweaver-Burk plots in two substrate kinetics experiments revealed both parallel line and intersecting initial velocity line patterns for deglutathionylation. Computational and mathematical models were used to resolve these discrepancies and we showed that the mono- and di- thiol mechanisms, are in fact identical. Mathematical models of mutant and wild-type glutaredoxin activities revealed that the GSH concentration and the rate constant for GSH oxidation significantly affected these relative activities which explained the contradictory data for wild-type and mutant glutaredoxins. The sigmoidal response to GSH was due to the kinetic order of this reaction and our results demonstrated that the resulting parallel and intersecting kinetic line patterns observed in some studies depended on the reversibility of the deglutathionylation reaction. Finally, fitting experiments showed that our models were able to accurately describe the in vitro data. Collectively, our results showed how deglutathionylation should be described in computational systems biology models and further revealed how the formation of oxidised glutaredoxin may play a vital role in the regulation of glutaredoxin activity.