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.
Akerman
Susan E
SE
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Müller
Sylke
S
D000818:Animals; D005786:Gene Expression Regulation; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054464:Peroxiredoxins; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000502:physiology; D021122:Protein Subunits; D015800:Protozoan Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013879:Thioredoxins / Q000378:metabolism
D021122:Protein Subunits; D015800:Protozoan Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D010544:Peroxidases; C477005:2-Cys peroxiredoxin, Plasmodium falciparum; D054464:Peroxiredoxins