Biochimica et Biophysica Acta (BBA) - General Subjects
ReviewCatalytic mechanisms and specificities of glutathione peroxidases: Variations of a basic scheme
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.
Section snippets
The kinetic mechanism of glutathione peroxidases and its basic chemistry
The prototype of glutathione peroxidases, mammalian GPx-1, catalyzes the reaction:ROOH + 2 GSH → GSSG + ROH + H2O
The kinetic mechanism of bovine GPx-1 was reported [26], [27] to correspond to the mechanism IV of the systematic of bimolecular reaction compiled by Dalziel and described as follows: “The enzyme reacts with each substrate in turn to form a product, itself undergoing intermediate chemical change, e. g. oxidation or reduction” [28]. Dalziel further distinguishes two subtypes of this
3. Activation of UP or CP
Basically, the oxidation of a thiolate or selenolate by H2O2, as a nucleophilic displacement reaction, is trivial. The still unsolved problem is how the incredible velocity of this reaction is achieved within the enzymes. Most commonly, the surprising velocity is explained by neighboring effects that force the peroxidatic cysteine (CP) or selenocysteine (UP) into dissociation. Similarly, the superiority of the seleno peroxidases is attributed to the lower pK of selenocysteine (5.2) versus
The chemical nature of F
So far the oxidized form of Se-GPx, F, has been presented as having the UP oxidized to its selenenic acid derivative. For reasons of stoichiometry we are, in fact, left without any known alternative for a reaction between one hydroperoxide with one selenol. Experimentally, however, the chemical nature of F has not yet been clarified. Compounds known to scavenge sulfenic acids such as dimedone and by analogy should react with selenenic acids do not inhibit GPx. Further, selenenic acids are
Hydroperoxide specificity
As mentioned, glutathione peroxidases are quite unspecific in respect to their oxidizing substrates. However, differences in specificity do exist and have important biological consequences (see below). GPx-1 had been shown to reduce a large variety of organic hydroperoxides including lipid hydroperoxides already in the late 60ies [6], [7]. It had therefore been implicated in the protection of biomembranes from lipid peroxidation, until two groups independently reported that the hydroperoxy
Physiological implications
The multiplicity of closely related enzymes in one species, of course raises questions: Are they simply backing up each other in an antioxidant network? Are the discrete differences in specificity physiologically relevant? What are the implications of their differential distribution in the organism? In this short overview, these problems cannot possibly be addressed for the entire GPx family. We will therefore restrict our discussion on some physiological aspects related to mammalian GPxs in
Conclusions and outlook
Since its discovery GPx-1 has attracted interest as the paradigm for antioxidant enzymes that link the most abundant cellular “antioxidant” GSH to hydroperoxide metabolism. Its selenoprotein nature seemed to explain its efficiency in hydroperoxide detoxification and contributed to the misconception that the essential trace element selenium is little else than a biological antioxidant. The focus of research was hardly changed when GPx-4, as the second family member, was demonstrated to catalyze
Acknowledgment
Authors wish to thank Walter de Gruyter for permitting to use the material for Fig. 9.
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