Permutation of the active site motif of tryparedoxin 2.
Tryparedoxins (TXN) are thioredoxin-related proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothione-dependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, 40WCPPCR45, of Crithidia fasciculata was mutated by site-directed mutagenesis and eight corresponding muteins were expressed in E. coli as terminally His-tagged 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 primarilythe 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.
Steinert
P
P
Department of Biochemistry, Technical University of Braunschweig, Germany.
Plank-Schumacher
K
K
Montemartini
M
M
Hecht
H J
HJ
Flohé
L
L
D000818:Animals; D001665:Binding Sites; D016831:Crithidia fasciculata; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
D015800:Protozoan Proteins; C114110:tryparedoxin; D013879:Thioredoxins