The evolved enzyme contains two mutations: L314H and D408E. The evolved enzyme and its parent homologue (GRLMR) were evaluated with the substrates oxidized glutathione (GSSG), selenodiglutathione, and selenite. Enzymatic reactions were started by addition of NADPH to a final concentration of 200 μM. Enzyme activity was quantified spectrophotometrically by observing depletion of the characteristic NADPH absorption maximum at 340 nm. The enzyme reactions were terminated upon observation of steady state reaction kinetics.