CATALYTIC CHARACTERISTICS OF TRYPAREDOXIN

Tryparedoxin, a thioredoxin-related protein from Crithidia fasciculata with a molecular mass of 16 kDa catalyses the reduction of a peroxire doxin-type peroxidase, Cf21, at the expense of trypanothione [Nogoceke , E., Gommel, D. U., Kie ss, M., Kalisz, H. M. & Flohe, L. E. (1997) B iol. Chem. Hoppe-Seyler 378, 827-836]. The kinetic analysis of trypare doxin revealed an enzyme substitution mechanism. The corresponding mol ecular event was elucidated to be a reversible oxidoreduction of the d isulfide bridge in the thioredoxin-related motif WCPPC. The amino-prox imal cysteine residue of this active site was more reactive in S-alkyl ation experiments than the distal residue. The natural substrates of t ryparedoxin, trypanothione and Cf21, could only be substituted by glut athione and glutathione disulfide with considerable loss in activity. The pronounced specificity of tryparedoxin is further accentuated by l ow limiting K-m values for Cf21 and trypanothione (2.2 mu M and 130 mu M, respectively, as compared to 990 mu M for gluthathione disulfide a nd an infinite value for glutathione). Tryparedoxin can therefore be c lassified as a trypanothione:peroxiredoxin oxidoreductase. The reducti on of tryparedoxin by trypanothione appears to be the rate-limiting st ep in the trypanothione-dependent hydroperoxide reduction because(a) t he regeneration of reduced tryparedoxin from the tryparedoxin-trypanot hione complex is rate limiting (k(cat) 392 min(-1)), (b) the physiolog ical trypanothione concentrations may not always saturate tryparedoxin , and (c) the rate constants for the net forward reaction of Cf21 are faster than those of the tryparedoxin reaction, The functional charact eristics of tryparedoxin explain the limited capacity of trypanosomati ds in coping with oxidative stress and qualify the enzyme as a potenti al target for the design of specific trypanocidal compounds.