Figures & data
Figure 1. Mechanism of catalysis of deglutathionylation by GRX. A glutathionylated protein (PS-SG) is restored by the action of RX. GRX operates by a monothiol mechanism in the process of deglutathionylation. The glutathione (GSH) is transferred from the protein to GRX. A GSH molecule restores the normal GRX active site structure with the production of GSSG. The GSSG produced is reduced to GSH by the action of GR which uses NADPH as a cofactor.
Figure 2. Structure of 2-AAPA.
Figure 3. Time and concentration dependence of GRX-1 inhibition by 2-AAPA. The natural logarithm of GRX-1 remaining activity is plotted against time. The enzyme was incubated with increasing concentrations of 2-AAPA, and aliquots were withdrawn at various time points for determination of remaining GRX-1 activity. The graph shows a representative plot from one of triplicate experiments. ▪, 200 μM; ▴, 100 μM; ×, 50 μM; •, 25 μM.
Figure 4. Double reciprocal plot. The reciprocals of the apparent rate constants of inhibition (slopes from ) are plotted against the reciprocals of 2-AAPA concentration. The graph shows a representative plot from one of triplicate experiments.
Figure 5. Irreversibility of GRX-1 inhibition by 2-AAPA. GRX-1 was incubated with 2-AAPA 1 mM for 1 hour to achieve complete inhibition. Then the solution was transferred to a DisPoDialyzer and dialyzed in Tris buffer for 4 hours. No enzyme activity returned in the 2-AAPA sample over a 4 hour period. The graph shows a representative plot from one of triplicate experiments. ♦, control; ▪, 2-AAPA treated.
Figure 6. Substrate protection of GRX from 2-AAPA inhibition. GRX substrate GSH-HED protected the enzyme from inhibition by 2-AAPA in a concentration dependent manner indicating that 2-AAPA is a competitive inhibitor of GRX-1 (n = 3).
Figure 7. (A) LC/MS analysis of covalent binding of 2-AAPA to GRX-1. GRX-1 inhibited by 2-AAPA 0.1 mM for 20 minutes. The native enzyme has an m/z of 11641. In the inhibited sample, additional peaks are observed at m/z 11,997 and 12,353, corresponding to monothiocarbamoylation at one or two cysteines, respectively. (B) LC/MS analysis of covalent binding of 2-AAPA to GRX. GRX-1 with 2-AAPA 0.1 mM and substrate (GSH 2 mM/HED 1.4 mM). The signals from the addition of 2-AAPA are not observed in the sample incubated with inhibitor and substrate; the signal at m/z 11,948 corresponds to the addition of glutathione.
Figure 8. Proposed mechanism of 2-AAPA binding to GRX-1. Data from mass spectrometry analysis of the inhibited enzyme show that 2-AAPA binds covalently to GRX-1. Monothiocarbamoylation of the enzyme can occur at one or two cysteines in GRX. Based on the pKa, it is likely that the monothiocarbamoylation leading to GRX-1 inhibition is occurring at CYS-22.
