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 , PfPrx6 , and PfPrx6 revealed a preference for H O as an oxidant with second order rate constants for H O and tBuOOH around 2.5 × 10 M s and 3 × 10 M s , respectively. Differences between the oxidation kinetics of PfPrx6 , PfPrx6 , and PfPrx6 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 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 might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.

Characterization of the glutathione‐dependent reduction of the peroxiredoxin 5 homolog PfAOP from Plasmodium falciparum

“…This is not a general feature of peroxiredoxins. For example, the reduction of peroxiredoxin 6‐type enzymes usually occurs very slowly in the presence of GSH or other common reducing agents 7,21,26–30 . Deciphering the underlying cause for the different reactivity will require much more structural and kinetic information.…”

“…For example, the reduction of peroxiredoxin 6-type enzymes usually occurs very slowly in the presence of GSH or other common reducing agents. 7,21,[26][27][28][29][30] Deciphering the underlying cause for the different reactivity will require much more structural and kinetic information. We therefore suggest to compare not only the structures but also the rate constants and thermodynamic activation parameters of alternative combinations of 1-Cys peroxiredoxins and thiol substrates to better understand this part of the reaction cycle.…”

Substrate Promiscuity and Hyperoxidation Susceptibility as Potential Driving Forces for the Co-evolution of Prx5-Type and Prx6-Type 1-Cys Peroxiredoxin Mechanisms

“…One major structural difference between Prx subfamilies is a residue at the bottom of the active site that probably affects the reactivity of the peroxidatic cysteine and the stability of the fully folded enzyme conformation. 7,13,19 Prx1-type enzymes usually have a tyrosine at this position, Prx5-type enzymes a hydrophobic residue, and Prx6-type enzymes a histidine residue (Figure 1C). 13,19,20 What are the benefits and drawbacks of a 1-Cys mechanism?…”

“…13 Recombinant PfPrx6 (also known as Pf1-Cys-Prx) exists in a monomer-dimer equilibrium 29 and was shown to rapidly react with hydroperoxides. 13 However, as is the case for many Prx6-type enzymes, 29−33 the physiological reducing agent of PfPrx6 remains unknown and the recombinant enzyme has only negligible peroxidase activity in steady-state kinetic assays with either thioredoxin 1 (PfTrx1) or GSH and glutaredoxin (PfGrx) as reducing agents. 13,29 Here, we compared the reaction mechanisms and kinetics of the Prx5-and Prx6-type 1-Cys Prx isoforms PfAOP and PfPrx6.…”

“…13 However, as is the case for many Prx6-type enzymes, 29−33 the physiological reducing agent of PfPrx6 remains unknown and the recombinant enzyme has only negligible peroxidase activity in steady-state kinetic assays with either thioredoxin 1 (PfTrx1) or GSH and glutaredoxin (PfGrx) as reducing agents. 13,29 Here, we compared the reaction mechanisms and kinetics of the Prx5-and Prx6-type 1-Cys Prx isoforms PfAOP and PfPrx6. We analyzed the substrate promiscuity of PfAOP and PfPrx6 using a variety of potential low and high-molecular weight reducing agents, determined missing rate constants for the catalytic cycle of PfAOP, and validated the generated kinetic models for substrate turnover and enzyme inactivation using partial and complete single turnover experiments as well as CD spectroscopy.…”