Activation of active-site cysteine residues in the peroxiredoxin-type tryparedoxin peroxidase of Crithidia fasciculata

Tryparedoxin peroxidase (TXNPx), recently identified as the hydroperoxide-d etoxifying enzyme of trypanosomatidae [Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M. & Flohe, L. (1997) Biol. Chem. 378, 827-836], is a member of the peroxiredoxin family and is characterized by two VCP motifs. Based on a consensus sequence of TXNPx and peroxiredoxin-type peroxidoses, eight TXNPx variants were designed, heterologously expressed in Escherichia coli, checked for BI-helix content by CD and kinetically analysed. The variant Q 164E was fully active, C52S, W87D and R128E were inactive and C173S, W87H, W177E and W177H showed reduced activity. Wild-type TXNPx and Q164E exhibit ping-pone kinetics with infinite maximum velocities, whereas saturation kin etics were observed with C173S and W177E. The data comply with a mechanism in which C52, primarily activated by R128 and possibly by W87, is first oxi dized by hydroperoxide to a sulfenic acid derivative. C173, supported by W1 77, then forms an intersubunit disulfide bridge with C52. If C173 is exchan ged with a redox-inactive residue (Ser) or is insufficiently activated, the redox shuttle remains restricted to C52. The shift in the kinetic pattern and decrease in specific activity of C173S and W177E may result from a limi ted accessibility of the oxidized C52 to tryparedoxin, which in the oxidize d wild-type TXNPx presumably attacks the C173 sulfur of the disulfide bridg e. The proposed mechanism of action of TXNPx is consistent with that deduce d for the homologous thioredoxin peroxidase of yeast [Chae, H. Z., Uhm, T. B. & Rhee. S. G. (1994) Proc. Natl Acad. Sci. USA 91, 7022-7026] and is sup ported by molecular modelling based on the structure of the human peroxired oxin 'hORF6' [Choi, H.-J., Kang, S. W. Yang, C.-H., Rheel S. G. Bi Ryu, S.- E. (1998) Nat. Struct. Biol. 5, 400-406].