Tryparedoxin peroxidase (TXNPx), recently identified as the hydroperoxide-detoxifying enzyme of trypanosomatidae [Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M. & Flohe Â, 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 peroxidases, eight TXNPx variants were designed, heterologously expressed in Escherichia coli, checked for a-helix content by CD and kinetically analysed. The variant Q164E 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-pong kinetics with infinite maximum velocities, whereas saturation kinetics 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 oxidized by hydroperoxide to a sulfenic acid derivative. C173, supported by W177, then forms an intersubunit disulfide bridge with C52. If C173 is exchanged 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 limited accessibility of the oxidized C52 to tryparedoxin, which in the oxidized wild-type TXNPx presumably attacks the C173 sulfur of the disulfide bridge. The proposed mechanism of action of TXNPx is consistent with that deduced for the homologous thioredoxin peroxidase of yeast Keywords: active site; Crithidia fasciculata; trypanothione; tryparedoxin peroxidase; tryparedoxin.Tryparedoxin peroxidase (TXNPx) has recently been discovered to be the terminal peroxidase of trypanosomal peroxide metabolism [1]. It requires tryparedoxin, a thioredoxin-related protein, as reducing substrate. Reduced tryparedoxin is in turn regenerated through direct reduction by trypanothione [N 1 ,N 8 -bis(glutathionyl)spermidine], a redox mediator typical of trypanosomatids. The reduction of oxidized trypanothione is achieved at the expense of NADPH by the flavoprotein trypanothione reductase (Fig. 1). This unique cascade of oxidoreductases has attracted considerable interest as a target for the design of trypanocidal drugs, as the proteins involved appear to be sufficiently distinct from any homologous or functionally analogous enzyme of the parasites' hosts [2±4].Chemically, TXNPx belongs to a family of antioxidant proteins named peroxiredoxins [5]. They comprise the alkylhydroperoxide reductases of bacteria [6], thioredoxin peroxidases of yeast and mammals [5] and a variety of proteins of unknown function [7,8]. Recently, the peroxiredoxin family has been subdivided into two groups, 1-Cys and 2-Cys peroxiredoxins, according to the number of conserved cysteines [9]. Typically, 2-Cys peroxiredoxins contain two VCP motifs. The VCP motif near the N-terminus is strictly conserved, whereas the second VCP motif appears to tolerate some sequence variation. For yeast thioredoxin peroxidase, ...

Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin-type peroxidases by the bis-glutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. The 3D-structures of the following tryparedoxins are presented: authentic tryparedoxin1 of Crithidia fasciculata, CfTXN1; the his-tagged recombinant protein, CfTXN1H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the active site motif 40-WCPPCR-45 proved to be the only solvent-exposed redox active residue in CfTXN2. In reduced TXNs, its nucleophilicity is increased by a network of hydrogen bonds. In oxidised TXNs it can be attacked by the thiol of the 1N-glutathionyl residue of trypanothione, as evidenced by the structure of 1N-glutathionylspermidine-derivatised CfTXN2H6-Cys44Ser. Modelling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cis-Pro111 (110)-bond and Arg129 (128) to be involved in the binding of trypanothione to CfTXN2 (CfTXN1). The model of TXN-substrate interaction is consistent with functional characteristics of known and newly designed muteins (CfTXN2H6-Arg129Asp and Glu73Arg) and the 1N-glutathionyl-spermidine binding in the CfTXN2H6-Cys44Ser structure.

Tryparedoxin peroxidase from

Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.