Probing the Presumed Catalytic Triad of Selenium-Containing Peroxidases by Mutational Analysis of Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx)

Abstract

Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis. The rate constants k+1 and k'+2, for the oxidation and the regeneration of the ground state enzyme were estimated, respectively. Moreover, the alkylation rate of the reactive centre by iodoacetate (kinact.) was also analysed. The substitution of the catalytically competent selenocysteine 46 by cysteine (PHGPxcys46) decreased k+1 and k'+2 by about three orders of magnitude, although leaving unaffected kinact.. Furthermore, mutations of PHGPxcys46 involving the other residues of the triad decreased both kinact. and k+1, thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of the nucleophilic cysteine thiol. In general, substitutions of Gln 81 or Trp 136 by acidic residues in PHGPxcys46 most dramatically depressed the k+1 values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in these positions allowed an intermediate dissociation and induced a corresponding reactivity of the thiol. Our data, for the first time, reveal that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidases.

A Comparative Study on the Hydroperoxide and Thiol Specificity of the Glutathione Peroxidase Family and Selenoprotein P

“…The substrate concentration and the reaction rate at each time interval (typically from 1 to 3 min) thus obtained were then fitted to the Dalziel equation, indicating a ping-pong mechanism as in the case of cGPx, eGPx, and PHGPx. The rate constants k 1 and k 2 Ј were calculated from the Dalziel coefficients as described previously (17,27). The first step is the oxidation of selenol anion (E-Se Ϫ ) by hydroperoxide to yield a selenenic acid derivative (E-SeOH) and hydroxide.…”

Molecular biology of glutathione peroxidase 4: from genomic structure to developmental expression and neural function

“…The catalytic selenocysteine residue at position 46 (U46) is essential and mutation of this amino acid to a cysteine (U46C) strongly impairs the catalytic efficiency (Maiorino et al, 1995;Schnurr et al, 2000). Kinetic analysis of the GPx reaction suggested a tert-uni ping pong mechanism, which involves redox shuttling of the selenocysteine (Ursini et al, 1982).…”

“…Structural models of GPx4 have been generated (Aumann et al, 1997;Mauri et al, 2003) based on the X-ray coordinates of other GPx isoforms (Epp et al, 1983;Ren et al, 1997). Although the degree of amino acid conservation among GPx isoforms is rather low, these models served in the past as suitable tools to predict target amino acids for site-directed mutagenesis to impede the catalytic activity (Maiorino et al, 1995(Maiorino et al, , 1998. In retrospect, this hypothetical model adequately mirrored the structural features of GPx4 as indicated by recent crystallographic data (Figure 4).…”

Induction and Functional Analysis of Two Reduced Nicotinamide Adenine Dinucleotide Phosphate-Dependent Glutathione Peroxidase-Like Proteins in Synechocystis PCC 6803 during the Progression of Oxidative Stress

“…GPX-like proteins of plants exhibit the highest identity to the mammalian selenium-dependent phospholipid hydroperoxide GPX. However, these plant genes carry a codon for Cys residues instead of selenocysteine residue in mammalian GPXs, resulting in lower activity by three orders of magnitude compared to the activity of the homologous animal GPX (Maiorino et al, 1995). These facts thus lead to a limited understanding of the potential physiological role of GPXlike proteins in photosynthetic organisms.…”