Catalytic mechanisms and specificities of glutathione peroxidases: Variations of a basic scheme

Abstract

Kinetics and molecular mechanisms of GPx-type enzymes are reviewed with emphasis on structural features relevant to efficiency and specificity. In Sec-GPxs the reaction takes place at a single redox centre with selenocysteine as redox-active residue (peroxidatic Sec, U_P). In contrast, most of the non-vertebrate GPx have the U_P replaced by a cysteine (peroxidatic Cys, C_P) and work with a second redox centre that contains a resolving cysteine (C_R). While the former type of enzymes is more or less specific for GSH, the latter are reduced by “redoxins”. The common denominator of the GPx family is the first redox centre comprising the (seleno)cysteine, tryptophan, asparagine and glutamine. In this architectural context the rate of hydroperoxide reduction by U_P or C_P, respectively, is enhanced by several orders of magnitude compared to that of free selenolate or thiolate. Mammalian GPx-1 dominates H₂O₂ metabolism, whereas the domain of GPx-4 is the reduction of lipid hydroperoxides with important consequences such as counteracting 12/15-lipoxygenase-induced apoptosis and regulation of inflammatory responses. Beyond, the degenerate GSH specificity of GPx-4 allows selenylation and oxidation to disulfides of protein thiols. Heterodimer formation of yeast GPx with a transcription factor is discussed as paradigm of a redox sensing that might also be valid in vertebrates.

Introduction

Glutathione peroxidases were the first seleno enzymes that were discovered in mammals [1], [2], [3], [4]. The “classical” glutathione peroxidase, now called GPx-1, was first described as an erythrocyte enzyme that specifically reduces H₂O₂ by GSH [5], but was later shown to also reduce a broad scope of organic hydroperoxides [6], [7]. Although it could protect biomembranes against spontaneous lipid peroxidation in mitochondrial membranes [8], which was likely mediated by H₂O₂, it did not reduce hydroperoxy groups of complex lipids [9], [10]. The latter activity could, however, be attributed to a second type of selenium-containing glutathione peroxidases that, in consequence, was termed “phospholipid hydroperoxide glutathione peroxidase” [4], now GPx-4. Glutathione peroxidase activity was also found to be associated with proteins that are not related in sequence with GPx such as glutathione S-transferase [11], selenoprotein P [12] and human peroxiredoxin 6 [13], but the term “glutathione peroxidase” (GPx) is now commonly used to define a family of phylogenetically related proteins, as is evidenced by sequence homology. This family has meanwhile grown up to more than 700 members spread over all domains of life [14], the majority only being known by DNA sequence. In mammals, up to 8 distinct glutathione peroxidases were detected. Most of them are selenoproteins (mammalian GPx-1, GPx-2, GPx-3, GPx-4 and, depending on species, GPx-6), while in the remaining 2 or 3 variants the active site selenocysteine residue is replaced by cysteine. Only GPx-1, 3 and 4 have been functionally characterized to some extent. Selenium-containing GPxs were also discovered in non-mammalian vertebrates such as fish [15] and birds [16], in parasitic trematodes [17] and sporadically in protists and bacteria [18]. The overwhelming majority of non-vertebrate GPxs, however, are cysteine homologues [19].

The participation of the selenocysteine residue in the catalysis of the GPxs was demonstrated by site-directed mutagenesis. Whenever the selenocysteine was exchanged to cysteine in a GPx, the specific activity dropped by two to three orders of magnitude [20], [21]. For long, therefore, it had been questioned whether the cysteine homologues could be peroxidases in the sense that they are able to catalyze hydroperoxide reduction at any physiologically relevant rate. More recently, however, kinetic analyses of naturally occurring Cys-GPxs revealed surprisingly high reaction rates. This unexpected efficiency had remained undetected until the real substrate of these “glutathione peroxidases” was discovered. First the GPx of Plasmodium falciparum [22], then several congeners of plant, yeast, protist and insect origin were shown to be reduced by “redoxins”, i.e. thioredoxins or related proteins with a CXXC motif, and, according to a recent bio-informatic prediction, this unexpected co-substrate specificity may be very common in GPxs of non-vertebrate taxa such as Alveolata, Arthropoda, Bacteria, Euglenozoa, Fungi and green plants [23]. In so far, the term “glutathione peroxidase” turned out to be a misnomer for most members of the GPx family. The redoxin-specific GPxs further surprised in mimicking peroxiredoxin catalysis in working with two catalytic redox-centers [23], [24], [25].

The present review will compile the accumulated knowledge on typical mammalian glutathione peroxidases with particular emphasis on their catalytic mechanism and the structural basis of specificities. The variations of the classical reaction scheme of non-vertebrate homologues will be presented and discussed in respect to possible roles of mammalian GPxs of still ill defined biological roles.