Abstract
Tryparedoxins (TXN) are thioredoxinrelated proteins
which, as trypanothione:peroxiredoxin oxidoreductases,
constitute the trypanothionedependent antioxidant
defense and may also serve as substrates for
ribonucleotide reductase in trypanosomatids. The active
site motif of TXN2, [40]WCPPCR[45], of Crithidia fasciculata was mutated by sitedirected mutagenesis and eight corresponding muteins were expressed in E. coli as terminally Histagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and,
if possible, kinetics. Exchange of Cys41 and Cys44 by
serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45
by aspartate resulted in loss of activity, suggesting
an activation of active site cysteines by the positive
charge of Arg45. Substitution of Trp40 by phenylalanine
or tyrosine resulted in moderate decrease of
specific activity, as did exchange of Pro42 by glycine.
Kinetic analysis of these three muteins revealed that
primarily the reaction with trypanothione is affected by
the mutations. Simulation of thioredoxin or glutaredoxin
like active sites in TXN2 (P42G and W40T/P43Y,
respectively) did not result in thioredoxin or glutaredoxin
like activities. These data underscore that
TXNs, although belonging to the thioredoxin superfamily,
represent a group of enzymes distinct from
thioredoxins and glutaredoxins in terms of specificity,
and appear attractive as molecular targets for the
design of trypanocidal compounds.