Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR)

Figure 5

Full time progress curves of T. crassiceps TGR as a function of both NADPH and GSSG concentration. Enzyme assays and incubation conditions were as described under Materials and Methods. The particular concentration of NADPH, GSSG, and enzyme used in the corresponding enzyme assays were as follows: Panel (a): (○) 4.2 μM NADPH, 5 μM GSSG, 14.7 nM TGR; (●) 4.7 μM, 270 μM GSSG, 11.5 nM TGR; (Δ) 4.9 μM NADPH, 500 μM GSSG, 11.5 nM TGR. Panel (b): (○) 10.1 μM NADPH, 20 μM GSSG, 14.7 nM TGR; (●) 13.9 μM NADPH, 200 μM GSSG, 11.5 nM TGR; (Δ) 13.4 μM NADPH, 510 μM GSSG, 11.5 nM TGR. Panel (c): (○) 47 μM NADPH, 120 μM GSSG, 11.5 nM TGR; (●) 46.5 μM NADPH, 200 μM GSSG, 11 nM TGR; (Δ) 46 μM NADPH, 310 μM GSSG, 6.6 nM TGR. Continuous lines represent fitting of experimental data points to the mechanism-based model using the best set of rate constants (see Materials and Methods for details of the conditions used in the fitting procedure).

(a)

(b)

(c)