2-Cys peroxiredoxin PfTrx-Px1 is involved in the antioxidant defence of Plasmodium falciparum.

Peroxiredoxins (Trx-Px) are ubiquitous antioxidant enzymes that catalyse the thioredoxin-dependent reduction of hydroperoxides. The number of characteristic active site (VCP/T) motifs defines these proteins as 1-Cys and 2-Cys Trx-Px. Steady-state kinetic parameters of Plasmodium falciparum 2-Cys Trx-Px (PfTrx-Px1) were determined using stopped flow rapid kinetics. The bi-substrate reaction displays ping-pong kinetics and the K(m) values for H2O2 and thioredoxin were determined to be 0.78+/-0.14 microM and 18.94+/-3.01 microM, respectively. The Vmax(app) and kcat(app) for H2O2 were found to be 4+/-0.6 U mg(-1) and 1.67+/-0.25 s(-1), respectively and those for thioredoxin are 23.0+/-0.2 U mg(-1) and 9.65+/-0.1 s(-1), emphasising the specificity of the enzyme for the substrate H2O2. After subjection to exogenous and endogenous oxidative stress, P. falciparum blood stage forms showed a marked elevation of PfTrx-Px1 mRNA and protein levels consistent with the hypothesis that it is an important component of the parasite's antioxidant machinery. Gel filtration, cross-linking and electron microscopy (EM) revealed that the protein forms decamers consisting of pentamers of homodimers that have a doughnut-like shape consistent with the structures of related proteins. No dimeric forms of the protein were detectable after gel filtration suggesting that PfTrx-Px1 predominantly exists as an oligomer.