In:
Protein Science, Wiley, Vol. 28, No. 1 ( 2019-01), p. 100-110
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 H 2 O 2 as an oxidant with second order rate constants for H 2 O 2 and tBuOOH around 2.5 × 10 7 M −1 s −1 and 3 × 10 6 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.
Type of Medium:
Online Resource
ISSN:
0961-8368
,
1469-896X
Language:
English
Publisher:
Wiley
Publication Date:
2019
detail.hit.zdb_id:
2000025-X
SSG:
12
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