CATALYTIC AND POTENTIOMETRIC CHARACTERIZATION OF E201D AND E201Q MUTANTS OF TRYPANOSOMA-CONGOLENSE TRYPANOTHIONE REDUCTASE

Trypanothione reductase is a member of the structurally and functional ly well-characterized family of flavoprotein reductases, which catalyz e the reduced pyridine nucleotide dependent reduction of their disulfi de, peroxide, or metal ion substrates. Trypanothione reductase is foun d in a wide variety of Trypanosoma species, where the enzyme serves ph ysiologically to protect the organism from oxidative stress and assist s in maintaining low intracellular levels of hydrogen peroxide. The re dox potential of the flavin and the hydride ion transfer reaction of t he pro-S hydrogen of NADPH to N5 of FAD have been proposed to be influ enced by the presence of a conserved Lys-Glu (K60-E201) ion pair at th e bottom of the nicotinamide binding pocket. We have evaluated this hy pothesis by making modest substitutions for both the Lys and Glu resid ues using site-directed mutagenesis. Replacement of the K60 residue wi th an arginine led to a poorly expressed, and completely inactive, enz yme. Replacement of the Glu201 residue with either a glutamine (E201Q) or an aspartate (E201D) residue led to expressed enzymes which could be readily purified in >20 mg amounts using protocols developed for th e WT enzyme, and which had significant residual trypanothione-reducing activity. These enzymes have now been characterized to determine thei r redox potentials, catalytic activities, and nucleotide specificities . Relative to the WT enzyme, both E201D and E201Q exhibit ca. 5% of WT trypanothione-reducing activity using NADPH as reductant, but signifi cantly enhanced quinone reductase activity. The oxidase activity of bo th mutants is enhanced by over 50-fold compared to that of the WT. The redox potential of the WT enzyme has been determined to be -273 mV, w hile both the E201D and E201Q exhibit more positive redox potentials ( -259 and -251 mV, respectively). These data confirm the modulating eff ect of the K60-E201 ion pair on the redox potential of the flavin, and the reactivity of the enzyme toward alternate reducible substrates.