The glutaredoxin mono- and di-thiol mechanisms for deglutathionylation are functionally equivalent: implications for redox systems biology

Abstract

Glutathionylation plays a central role in cellular redox regulation and anti-oxidative defence. Grx (Glutaredoxins) are primarily responsible for reversing glutathionylation and their activity therefore affects a range of cellular processes, making them prime candidates for computational systems biology studies. However, two distinct kinetic mechanisms involving either one (monothiol) or both (dithiol) active-site cysteines have been proposed for their deglutathionylation activity and initial studies predicted that computational models based on either of these mechanisms will have different structural and kinetic properties. Further, a number of other discrepancies including the relative activity of active-site mutants and contrasting reciprocal plot kinetics have also been reported for these redoxins. Using kinetic modelling, we show that the dithiol and monothiol mechanisms are identical and, we were also able to explain much of the discrepant data found within the literature on Grx activity and kinetics. Moreover, our results have revealed how an apparently futile side-reaction in the monothiol mechanism may play a significant role in regulating Grx activity in vivo.

Figure 1. A comparison of the Grx dithiol and monothiol mechanisms for deglutathionylation

In the dithiol mechanism (A), the N-terminal active-site cysteine of glutaredoxin (Grx(SH)₂) initiates a nucleophilic attack on the glutathionylated protein substrate (PSSG) resulting in a mixed disulphide that is attacked by the second C-terminal active-site cysteine releasing the reduced protein (protein SH) and GrxSS (reaction 2) which is subsequently reduced by two GSH molecules (reaction 3). In the monothiol mechanism (B), the N-terminal thiolate anion forms a GrxSSGSH releasing the reduced protein (reaction 2). This mixed disulphide is reduced by glutathione, regenerating active Grx (reaction 3). A side-reaction resulting in the formation of GrxSS detracts from catalysis. (reaction 4). GSSG is regenerated by glutathione reductase in both mechanisms (reaction 1).

Figure 2. The Grx dithiol and monothiol mechanisms for deglutathionylation are identical

Once the the glutaredoxin-GSH mixed disulfide (GrxSSGSH) intermediate is included in the dithiol mechanism (A) and oxidized glutaredoxin (GrxSS) formation is considered part of the monothiol mechanism (B), then the reaction schemes for these mechanisms are identical.

Figure 3. Kinetic models based on the Grx dithiol mechanism can successfully describe in vitro datasets

Yeast Grx dithiol models were fitted to datasets describing the reduction of HED by Grx 1 (A) and Grx 2 (B) [26]. For the Grx 1 dataset (A), the fitted rate constants for glutaredoxin and HED reduction were 4.23±0.30×10⁻⁶ μM⁻²·s⁻¹ and 0.073±0.011 μM⁻¹·s⁻¹ respectively, and the goodness of fit was assessed by an r² value of 0.94. Rate constants for glutaredoxin and HED reduction of 6.74±0.96×10⁻⁵ μM⁻²·s⁻¹ and 0.252±0.045 μM⁻¹·s⁻¹ respectively, were obtained for the Grx2 dataset with an r² value of 0.97.

Figure 4. The contrasting reciprocal plot kinetics for Grx substrates can be explained using a Grx dithiol computational model

Analysis of a validated E. coli glutaredoxin model [20] revealed that double-reciprocal plots for a glutathionylated substrate (PSSG) (A) or GSH (B) were expected to show distinct responses to changes in substrate concentration. In (A) the PSSG concentration was varied from 0.1–20 μM with a constant GSH concentration (1.0 mM) while in (B) the GSH concentration was varied from 0.1–4.0 mM with a constant PSSG concentration (5 μM).

Figure 5. Double reciprocal plots of the Grx system can show ping-pong or sequential kinetic patterns depending on the reversibility of the deglutathionylation reaction

An E. coli Grx computational model [20] with the deglutathionylation of a substrate (PSSG) modelled with irreversible (solid) or reversible mass action kinetics (dashes) at varying GSH concentrations of 150 (blue), 250 (red) and 1000 (black) μM was analysed (A). Reciprocal plots of the models revealed a ping-pong kinetic pattern (B) when deglutathionylation was modelled with irreversible kinetics but a sequential pattern was obtained when this reaction was modelled with reversible kinetics (C).

Figure 6. The formation of GrxSS may be a mechanism to prevent deglutathionylation in the presence of ROS

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