Figure 5.

(A) The plot monitors the consumption of NADPH by following the decrease in A₃₄₀. When 10 mM H₂O₂ is added to a reaction containing 50 nM mTR-GCUG and NADPH, mTR reduces H₂O₂ to water and becomes oxidized. The oxidized mTR is reduced by NADPH resulting in a decrease in A₃₄₀. After prolonged exposure (20 min) to excess H₂O₂, 90 μM Trx is added to the cuvette and the sharp increase in NADPH consumption (shown by a larger negative slope) shows that Trx is rapidly reduced and that a large excess of H₂O₂ does not inhibit the enzyme. (B) Comparison of activity progress curves for mTR treated with H₂O₂ (blue = 50 mM, green = 1 mM) and control mTR (red = no H₂O₂). After approximately 25 min, all of the NADPH in the reaction is consumed (for the enzyme treated with 50 mM H₂O₂) shown by a plateau in the slope. The samples were then treated with 14 units of catalase to remove excess H₂O₂ for 12 min. During this quenching step the A₃₄₀ was not monitored, but we have added a line to the plot for continuity. Since all of the NADPH was consumed in the sample treated with 50 mM H₂O₂, an additional bolus of NADPH was then added to the reaction to achieve a final concentration of 200 μM. The reaction was then monitored for 2 additional min at 340 nm to ensure all of the H₂O₂ was removed and then 90 μM Trx was added to each sample. The activity progress curves are extremely similar, even for the sample treated with 50 mM H₂O₂. The overall results show that mTR is very resistant to inactivation, even though the ability of the enzyme to turnover H₂O₂ shows that the Se-atom must be exposed to the oxidant.