Substrate and inhibitor specificities differ between human cytosolic and mitochondrial thioredoxin reductases: Implications for development of specific inhibitors.

Western Australian Institute for Medical Research, Centre for Medical Research, University of Western Australia, Perth, WA 6000, Australia.
Free Radical Biology & Medicine (Impact Factor: 5.27). 03/2011; 50(6):689-99. DOI: 10.1016/j.freeradbiomed.2010.12.015
Source: PubMed

ABSTRACT The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.

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    ABSTRACT: Cytosolic thioredoxin reductase 1 is the best characterized of the class of high molecular weight (Mr) thioredoxin reductases (TRs). TR1 is highly dependent upon the rare amino acid selenocysteine (Sec) for the reduction of thioredoxin (Trx) and a host of small molecule substrates, as mutation of Sec to cysteine (Cys) results in large drop in catalytic activity for all substrate types. Previous work in our lab and others has shown that the mitochondrial TR (TR3) is much less dependent upon the use of Sec for the reduction of small molecules. Sec-dependent substrate utilization behavior of TR1 may be the exception and not the rule as we show that a variety of high Mr) TRs from other organisms including D. melanogaster, C. elegans, and P. falciparum do not require Sec to reduce small molecule substrates including 5,5'dithio-bis(2-nitrobenzoic acid), lipoic acid, selenite, and selenocystine. The data shows that high Mr) TRs can be divided into two groups based upon substrate utilization patterns: a TR1 group and a TR3-like group. We have constructed mutants of TR3-like enzymes from mouse, D. melanogaster, C. elegans, and P. falciparum and the kinetic data from these mutants show that these enzymes are less dependent upon the use of Sec for the reduction of substrates. We posit that the mechanistic differences between TR1 and the TR3-like enzymes in this study are due to the presence of a "guiding bar", amino acids 407-422, found in TR1, but not TR3-like enzymes. The guiding bar, proposed by Becker and coworkers [Fritz-Wolf, K., Urig, S., and Becker, K. (2007) The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis, J Mol Biol 370, 116-127.], restricts the motion of the C-terminal tail containing the C-terminal Gly-Cys-Sec-Gly, redox-active tetrapeptide so that only this C-terminal redox center can be reduced by the N-terminal redox center, with the exclusion of most other substrates. This makes TR1 highly dependent upon the use of Sec because the selenium atom is responsible for both accepting electrons from the N-terminal redox center and donating them to the substrate in this model. Loss of both Se-electrophilicity and Se-nucleophilicity in the Sec → Cys mutant of TR1 greatly reduces catalytic activity. TR3-like enzymes in contrast are less dependent upon the use of Sec because the absence of the guiding bar in these enzymes allows for greater substrate access to the N-terminal redox center, and because they can make use of alternative mechanistic pathways that are not available to TR1.
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    ABSTRACT: Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redoxactive tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions by being: (i) a better nucleophile than Cys, (ii) a better electrophile than Cys, (iii) a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys<sub>497</sub>-Sec<sub>498</sub> dyad of the C-terminal redox center was mutated to either a Ser<sub>497</sub>-Cys<sub>498</sub> dyad or a <sub>Cys497</sub>-Ser</sub>498</sub> dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) in order to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser<sub>497</sub>-Cys<sub>498</sub> dyad formed a complex and this structure has been solved by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex, Nat Commun 2, 383.] This experimental observation most likely means that the selenolate is the initial attacking nucleophile onto the disulfide bond of Trx because a complex only resulted when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the 8-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could accelerate this exchange reaction by being a good leaving group (attack at the sulfur atom), or by being a good electrophile (attack at the selenium atom). Here we provide strong evidence that the selenium atom is attacked in this exchange step. This was shown by creating a mutant enzyme containing a Gly-Gly-Sec<sub>coo-</sub> motif that had 0.5% of the activity of the wild type enzyme. This mutant lacks the adjacent, resolving Cys residue, which acts by attacking the mixed selenosulfide bond that occurs between enzyme and substrate. A similar result was obtained when Sec was replaced with homocysteine. These results highlight the role of selenium as an electron acceptor in the catalytic mechanism of thioredoxin reductase as well as its established role as an electron donor to the substrate.
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May 22, 2014