Permutation of the active site motif of tryparedoxin 2

Tryparedoxins (TXN) are thioredoxin-related proteins which, as trypanothion e:peroxiredoxin oxidoreductases, constitute the trypanothione-dependent ant ioxidant defense and may also serve as substrates for ribonucleotide reduct ase in trypanosomatids, The active site motif of TXN2, (WCPPCR45)-W-40, of Crithidia fasciculata was mutated by site-directed mutagenesis and eight co rresponding muteins were expressed in E. coli as terminally His-tagged prot eins, purified to homogeneity by nickel chelate chromatography, and charact erized in terms of specific activity, specificity end, if possible, kinetic s. Exchange of Cys41 and Cys44 by serine yielded inactive products confirmi ng their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cyste ines by the positive charge of Arg45. Substitution of Trp40 by phenylalanin e or tyrosine resulted in moderate decrease of specific activity, as did ex change of Pro42 by glycine. Kinetic analysis of these three muteins reveale d that primarily the reaction with trypanothione is affected by the mutatio ns. Simulation of thioredoxin or glutaredoxin-like active sites in TXN2 (P4 2G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredox in-like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thi oredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.