Tyrosine substitution of a conserved active-site histidine residue activates Plasmodium falciparum peroxiredoxin 6

Abstract

Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro- and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so-called Prx6-type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady-state kinetics with tert-butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four- to six-fold in vitro. Stopped-flow kinetics with reduced PfPrx6^WT , PfPrx6^C128A , and PfPrx6^H39Y revealed a preference for H₂ O₂ as an oxidant with second order rate constants for H₂ O₂ and tBuOOH around 2.5 × 10⁷ M^-1 s^-1 and 3 × 10⁶ M^-1 s^-1 , respectively. Differences between the oxidation kinetics of PfPrx6^WT , PfPrx6^C128A , and PfPrx6^H39Y were observed during a slower second-reaction phase. Our kinetic data support the interpretation that the reductive half-reaction is the rate-limiting step for PfPrx6 catalysis in steady-state measurements. Whether the increased activity of PfPrx6^H39Y is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6-type enzymes is non-essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain-of-function mutant PfPrx6^H39Y might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.

Keywords: 1-Cys Prx; Plasmodium falciparum; Prx6; enzyme mechanism; peroxiredoxin catalysis; steady-state kinetics; stopped-flow kinetics.