A hypothesis on the catalytic mechanism of the selenoenzyme thioredoxin reductase

Gromer, Stephan; Wissing, Joseph; Behne, D.; Ashman, Keith; Schirmer, R. Heiner; Flohé, Leopold; Becker, Katja

doi:10.1042/bj3320591

Cited by 96 publications

(79 citation statements)

References 13 publications

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“…Most importantly, the pH optimum of the mutant enzyme was 9 as opposed to 7 for the wild-type selenium-containing enzyme compatible with the involvement of a low pK a (5.25) selenol in the catalytic mechanism (10). Accumulated evidence showed that the native unreduced enzyme was resistant to either carboxypeptidase (12) or trypsin digestion (36) as well as to chemical modification by 1-chloro-2,4-dinitrobenzene (22), aurothioglucose (37), bromo [1-14 C] acetic acid (20), and 5-iodoacetamidofluorescein (32). However, the enzyme was modified by all of the above treatments only after NADPH reduction.…”

Section: Discussionmentioning

confidence: 97%

Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence

Zhong

Arnér

Holmgren

2000

Proc. Natl. Acad. Sci. U.S.A.

452

343

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Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to glutathione reductase (GR), with an essential selenocysteine (SeCys) residue in an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. In the oxidized enzyme, we demonstrated two nonflavin redox centers by chemical modification and peptide sequencing: one was a disulfide within the sequence -Cys 59 -Val-Asn-Val-Gly-Cys 64 , identical to the active site of GR; the other was a selenenylsulfide formed from Cys 497 -SeCys 498 and confirmed by mass spectrometry. In the NADPH reduced enzyme, these centers were present as a dithiol and a selenolthiol, respectively. Based on the structure of GR, we propose that in TrxR, the C-terminal Cys 497 -SeCys 498 residues of one monomer are adjacent to the Cys 59 and Cys 64 residues of the second monomer. The reductive half-reaction of TrxR is similar to that of GR followed by exchange from the nascent Cys 59 and Cys 64 dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. Characterization of recombinant mutant rat TrxR with SeCys 498 replaced by Cys having a 100-fold lower kcat for Trx reduction revealed the C-terminal redox center was present as a dithiol when the Cys 59 -Cys 64 was a disulfide, demonstrating that the selenium atom with its larger radius is critical for formation of the unique selenenylsulfide. Spectroscopic redox titrations with dithionite or NADPH were consistent with the structure model. Mechanisms of TrxR in reduction of Trx and hydroperoxides have been postulated and are compatible with known enzyme activities and the effects of inhibitors, like goldthioglucose and 1-chloro-2,4-dinitrobenzene.evolution ͉ thiols ͉ disulfide ͉ selenium ͉ enzyme structure

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Section: Discussionmentioning

confidence: 97%

Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence

Zhong

Arnér

Holmgren

2000

Proc. Natl. Acad. Sci. U.S.A.

452

343

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“…The C-terminal redox-active motif in one subunit of the dimeric high M r TrxR enzymes receives electrons from two N-terminal redox-active cysteines proximal to an FAD in the other subunit, subsequently transferring these electrons to Trx or other substrates of the enzyme (7,8,11,21). By using stopped-flow kinetic methods, the rates of these separate events can be determined.…”

Section: Resultsmentioning

confidence: 99%

“…The catalytic activities of mammalian TrxR isoenzymes depend on a redox-active Cys-Sec couple within the C-terminal tetrapeptide motif, -GlyCys-Sec-Gly-COOH (4)(5)(6). Compared with sulfur, selenium is not only generally more reactive but also exhibits an Ϸ15% longer bond length, which facilitates formation of a selenenylsulfide bridge between the adjacent Cys-Sec residues, which is a necessary intermediate in the catalytic cycle (7)(8)(9)(10)(11). Mutational studies of the mammalian enzyme in which Sec was replaced by Cys showed a marked decrease in the catalytic rate (k cat ) for Trx reduction (10)(11)(12).…”

mentioning

confidence: 99%

Active sites of thioredoxin reductases: Why selenoproteins?

Gromer

Johansson

Bauer

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

196

192

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Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -SerCys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

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“…In the majority of known mammalian selenoproteins, selenium occurs in the form of selenocysteine, which has proved to be essential for efficient catalysis Gromer et al, 1998;Köhrle, 2000b;Lee et al, 2000). If selenocysteine is substituted by cysteine, the activity of the selenoenzymes falls by 2 to 3 orders of magnitude.…”

Section: Identified Selenoproteinsmentioning

confidence: 99%

“…It needs selenocysteine as the penultimate amino acid residue for its appropriate enzymatic function (Marcocci et al, 1997;Gromer et al, 1998;Lee et al, 2000;Gorlatov and Stadtman, 2000). Recently, two more tissue-specifically expressed isoenzymes were also identified as selenoproteins (Gasdaska et al, 1999;Lee et al, 1999;Watabe et al, 1999) and related proteins were identified by 'in silico' cloning (Lescure et al, 1999).…”

Section: Metabolic Function Of Mammalian Selenoproteinsmentioning

confidence: 99%

Selenium in Biology: Facts and Medical Perspectives

Köhrl¹,

Brigelius‐Flohé²,

Böck³

et al. 2000

Biological Chemistry

247

143

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Several decades after the discovery of selenium as an essential trace element in vertebrates approximately 20 eukaryotic and more than 15 prokaryotic selenoproteins containing the 21 st proteinogenic amino acid, selenocysteine, have been identified, partially characterized or cloned from several species. Many of these proteins are involved in redox reactions with selenocysteine acting as an essential component of the catalytic cycle. Enzyme activities have been assigned to the glutathione peroxidase family, to the thioredoxin reductases, which were recently identified as selenoproteins, to the iodothyronine deiodinases, which metabolize thyroid hormones, and to the selenophosphate synthetase 2, which is involved in selenoprotein biosynthesis. Prokaryotic selenoproteins catalyze redox reactions and formation of selenoethers in (stress-induced) metabolism and energy production of E. coli, of the clostridial cluster XI and of other prokaryotes. Apart from the specific and complex biosynthesis of selenocysteine, selenium also reversibly binds to proteins, is incorporated into selenomethionine in bacteria, yeast and higher plants, or posttranslationally modifies a catalytically essential cysteine residue of CO dehydrogenase. Expression of individual eukaryotic selenoproteins exhibits high tissue specificity, depends on selenium availability, in some cases is regulated by hormones, and if impaired contributes to several pathological conditions. Disturbance of selenoprotein expression or function is associated with deficiency syndromes (Keshan and Kashin-Beck disease), might contribute to tumorigenesis and atherosclerosis, is altered in several bacterial and viral infections, and leads to infertility in male rodents.

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A hypothesis on the catalytic mechanism of the selenoenzyme thioredoxin reductase

Cited by 96 publications

References 13 publications

Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence

Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence

Active sites of thioredoxin reductases: Why selenoproteins?

Selenium in Biology: Facts and Medical Perspectives

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