Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30044 (antioxidant enzyme)
 8,037 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Selenosubtilisin, a semisynthetic enzyme produced by chemical modification of subtilisin's catalytic serine, mimics the antioxidant enzyme glutathione peroxidase, catalyzing the reduction of hydroperoxides by 3-carboxy-4-nitrobenzenethiol. In analogy with the unmodified protease, selenosubtilisins derived from distantly related subtilisin templates exhibit significantly different kinetic properties. Selenosubtilisin BPN' not only is less active than the previously studied Carlsberg selenoenzyme but exhibits sequential rather than ping-pong kinetics, indicating the formation of a ternary complex between enzyme, thiol, and peroxide prior to product release. Experiments with subtilisin E and the BPN' Y217L variant show that the observed differences in kinetic mechanism and chemical efficiency can be attributed largely to amino acid substitutions in the enzyme's S1 and S1' binding sites, respectively. These contributions appear to be roughly additive, and a BPN' triple mutant (E156S/G169A/Y217L) has properties that closely approximate those of selenosubtilisin Carlsberg. The kinetic mechanism of selenosubtilisin can thus be controlled by limited mutagenesis of several active site residues not directly involved in the redox chemistry.
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PMID:Nonessential active site residues modulate selenosubtilisin's kinetic mechanism. 775 93

The crystal structures of three forms of Escherichia coli thioredoxin reductase have been refined: the oxidized form of the wild-type enzyme at 2.1 A resolution, a variant containing a cysteine to serine mutation at the active site (Cys138Ser) at 2.0 A resolution, and a complex of this variant with nicotinamide adenine dinucleotide phosphate (NADP+) at 2.3 A resolution. The enzyme mechanism involves the transfer of reducing equivalents from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to a disulfide bond in the enzyme, via a flavin adenine dinucleotide (FAD). Thioredoxin reductase contains FAD and NADPH binding domains that are structurally similar to the corresponding domains of the related enzyme glutathione reductase. The relative orientation of these domains is, however, very different in the two enzymes: when the FAD domains of thioredoxin and glutathione reductases are superimposed, the NADPH domain of one is rotated by 66 degrees with respect to the other. The observed binding mode of NADP+ in thioredoxin reductase is non-productive in that the nicotinamide ring is more than 17 A from the flavin ring system. While in glutathione reductase the redox active disulfide is located in the FAD domain, in thioredoxin reductase it is in the NADPH domain and is part of a four-residue sequence (Cys-Ala-Thr-Cys) that is close in structure to the corresponding region of thioredoxin (Cys-Gly-Pro-Cys), with a root-mean-square deviation of 0.22 A for atoms in the disulfide bonded ring. There are no significant conformational differences between the structure of the wild-type enzyme and that of the Cys138Ser mutant, except that a disulfide bond is not present in the latter. The disulfide bond is positioned productively in this conformation of the enzyme, i.e. it stacks against the flavin ring system in a position that would facilitate its reduction by the flavin. However, the cysteine residues are relatively inaccessible for interaction with the substrate, thioredoxin. These results suggest that thioredoxin reductase must undergo conformational changes during enzyme catalysis. All three structures reported here are for the same conformation of the enzyme and no direct evidence is available as yet for such conformational changes. The simplest possibility is that the NADPH domain rotates between the conformation observed here and an orientation similar to that seen in glutathione reductase. This would alternately place the nicotinamide ring and the disulfide bond near the flavin ring, and expose the cysteine residues for reaction with thioredoxin in the hypothetical conformation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Crystal structure of Escherichia coli thioredoxin reductase refined at 2 A resolution. Implications for a large conformational change during catalysis. 811 95

Selenosubtilisin, a semisynthetic enzyme produced by chemical modification of subtilisin's catalytic serine, mimics the antioxidant enzyme glutathione peroxidase, catalyzing the reduction of hydroperoxides by 3-carboxy-4-nitrobenzenethiol. In analogy with the natural peroxidase, a variety of hydroperoxides are accepted as substrates for the semisynthetic enzyme, whereas the dialkyl compound tert-butyl peroxide is not. Kinetic investigations reveal that kmax is dependent upon the nature of the hydroperoxide, indicating that peroxide-mediated oxidation of the enzymic selenolate is at least partially rate-limiting. Experiments with the radical trap 2,6-di-tert-butyl-4-methylphenol suggest that, while the nonenzymic reaction between tert-butyl hydroperoxide and thiol involves free radicals, the same reaction catalyzed by selenosubtilisin does not. The studies described here support the enzyme's proposed ping-pong mechanism and are consistent with previous mechanistic observations.
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PMID:Peroxide dependence of the semisynthetic enzyme selenosubtilisin. 826 74

Spermatozoa and seminal plasma from cockerels at the beginning and end of their reproductive periods were examined for their lipid composition and associated antioxidant capacities. The significant reduction in concentration of spermatozoa with age was associated with a large increase in lipid concentrations in spermatozoa and in seminal plasma. This change in lipid concentration was accompanied by increases in the proportions of phospholipid and free cholesterol; in contrast, the proportions of these lipid moieties in seminal plasma were reduced. The major phospholipid fractions in the spermatozoa and seminal plasma were phosphatidyl choline and phosphatidyl ethanolamine. There was a large decrease with age in the proportion of phosphatidyl ethanolamine and a commensurate increase in that of phosphatidyl choline in the spermatozoa and seminal plasma. These major changes in phospholipid content were accompanied by a decrease in the amount of phosphatidyl serine in the spermatozoa and increases in phosphatidyl inositol and cardiolipin in both spermatozoa and seminal plasma. The reductions in the proportions of phosphatidyl ethanolamine were accompanied by considerable reductions in the content of the major polyunsaturated fatty acids 20:4 (n-6) and 22:4 (n-6). The changes in lipid composition owing to ageing were associated with a marked reduction within the spermatozoa of the major antioxidant enzyme glutathione peroxidase. The role of these changes in the specific combinations of polyunsaturated lipids and in antioxidant capacity in the reduction in fertility with age are discussed.
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PMID:Lipid and antioxidant changes in semen of broiler fowl from 25 to 60 weeks of age. 869 2

Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice. In this work we report that while wild-type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an 'open', calcium-activated conformation, and that G93A-SOD1 must have its redox-active copper site available to substrates in order to exert its pro-oxidant properties on calcineurin. These findings demonstrate that both wild-type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin-regulated biochemical pathways in the pathogenesis of FALS.
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PMID:Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis. 1170 56

Depending on the availability of extracellular nutrients, yeast can enter either high or low metabolism survival phases. We have identified two pathways that regulate longevity and stress resistance in both the low and high metabolism phases. The deletion of SCH9, which encodes for a serine threonine kinase, triples the mean life span and increases resistance to oxidative and thermal stress. Mutations that decrease the activity of the Ras/Cyr1/PKA pathway also extend longevity and increase stress resistance by activating transcription factors Msn2/Msn4 and the mitochondrial antioxidant enzyme superoxide dismutase (Sod2). Although only one intracellular pathway that includes genes homologous to SCH9 and SOD2 has been identified in worms, our studies in yeast suggest that longevity in higher eukaryotes may also be negatively regulated by the Ras pathway.
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PMID:The Ras and Sch9 pathways regulate stress resistance and longevity. 1285 92

Hydrogen peroxide (H2O2), a reactive oxygen species, is assumed to have a detrimental effect on neuronal plasticity. Indeed, H2O2 suppresses long-term potentiation (LTP) in hippocampal slices of normal rats and wild-type (wt) mice. Transgenic mice overexpressing superoxide dismutase (SOD) 1 (tg-SOD), which maintain high ambient H2O2, have also been shown to be impaired in their ability to express hippocampal LTP. Paradoxically, H2O2, at a concentration (50 microm) that blocks LTP in wt mice, actually enhanced LTP in slices of 2-month-old tg-SOD mice. H2O2-dependent LTP in tg-SOD was blocked by the protein phosphatase calcineurin inhibitor FK506, but not by rapamycin, an FK-binding protein 12 (FKBP12) inhibitor or by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), a serine-kinase inhibitor. Interestingly, wt and tg-SOD mice expressed similar levels of the antioxidant enzyme catalase and similar activity of glutathione peroxidase. An opposite situation was found in 2-year-old mice. Aged wt mice were impaired in LTP in a manner that could be reversed by the addition of H2O2. Surprisingly, aged tg-SOD mice exhibited larger LTP than that found in wt mice, but this was now reduced by 50 microm H2O2. Both young tg-SOD and aged control mice displayed altered protein phosphatase activity, compared with that of young controls; moreover, FK506 inhibited LTP in old tg-SOD as well as in old wt mice treated with H2O2. These data promoted a dual role for H2O2 in the regulation of LTP, and proposed that it is mediated by the protein phosphatase calcineurin.
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PMID:Paradoxical actions of hydrogen peroxide on long-term potentiation in transgenic superoxide dismutase-1 mice. 1461 95

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a kcat of 610 min(-1) and a Km of 610 microM using E. coli thioredoxin as substrate. The reported kcat is 25% of the kcat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.
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PMID:Characterization of mitochondrial thioredoxin reductase from C. elegans. 1678 Jul 99

To investigate low-dose/low-dose-rate effects of low-linear energy transfer (LET) ionizing radiation, we used gamma-irradiated cells adapted to grow in a three-dimensional architecture that mimics cell growth in vivo. We determined the cellular, molecular and biochemical changes in these cells. Quiescent normal human fibroblasts were irradiated with single acute or chronic doses (1-10 cGy) of (137)Cs gamma rays. Whereas exposure to an acute dose of 10 cGy increased micronucleus formation, protraction of the dose over 48 h reduced micronucleus frequency to a level similar to or lower than what occurs spontaneously. The protracted treatment also up-regulated the cellular content of the antioxidant glutathione. These changes correlated with modulation of phospho-TP53 (serine 15), a stress marker that was regulated by doses as low as 1 cGy. The DNA damage that occurred after exposure to an acute dose of 10 cGy was protected against in two ways: (1) up-regulation of cellular antioxidant enzyme activity by ectopic overexpression of MnSOD, catalase or glutathione peroxidase, and (2) inhibition of superoxide anion generation by flavin-containing oxidases. These results support a significant role for oxidative metabolism in mediating low-dose radiation effects and demonstrate that cell culture in three dimensions is ideal to investigate radiation-induced adaptive responses. Expression of connexin 43, a constitutive protein of gap junctions, and the G(1) checkpoint were more sensitive to regulation by gamma rays in cells maintained in a three-dimensional than in a two-dimensional configuration.
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PMID:Adaptive responses to low-dose/low-dose-rate gamma rays in normal human fibroblasts: the role of growth architecture and oxidative metabolism. 1714 77

Peroxiredoxin I (Prx I) is an antioxidant enzyme with thioredoxin-dependent peroxidase activity which is involved in various cellular processes such as regulation of cell proliferation. Here, it is shown that the proinflammatory mediator lipopolysaccharide (LPS) inhibits the induction of Prx I expression and promoter activity by the phorbol ester 12-O-tetradecanoylphorbol- 13-acetate (TPA) in RAW264.7 monocytes, but not that of cyclooxygenase-2. LPS-dependent repression of Prx I induction by TPA was mediated via a newly identified kappaB site in the Prx I promoter, but the "classical" NF-kappaB cascade was not involved in this regulatory pathway, because IkappaB did not affect LPS-mediated Prx I repression. By contrast, phosphorylation of p65 at serine 276, which enhances the transcriptional activity of NF-kappaB, was up-regulated by TPA and was reduced by simultaneous exposure to LPS. Functional studies with Gal4-p65 constructs revealed that serine 276 is crucial to confer LPS-dependent repression of TPA-mediated induction of p65 transactivation. Finally, repression of TPA-dependent Prx I induction by LPS was mediated via Bruton's tyrosine kinase as indicated by studies with the pharmacological inhibitor LFM-A13. In summary, LPS-dependent inhibition of Prx I gene activation by TPA in monocytes is regulated via a pathway that involves phosphorylation of the NF-kappaB subunit p65 at serine 276.
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PMID:Inhibition of phorbol ester-dependent peroxiredoxin I gene activation by lipopolysaccharide via phosphorylation of RelA/p65 at serine 276 in monocytes. 1807 Jun 9


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