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Synthesis of peptide substrates for mammalian thioredoxin reductase.

ABSTRACT: Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.

SUBMITTER: Flemer S

PROVIDER: S-EPMC3690199 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Synthesis of peptide substrates for mammalian thioredoxin reductase.

Flemer Stevenson S Lacey Brian M BM Hondal Robert J RJ

Journal of peptide science : an official publication of the European Peptide Society 20080501 5

Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We dev ...[more]

PMID: 18035847

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Project description:Cytosolic thioredoxin reductase 1 (TR1) 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 a large decrease 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. The 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 Drosophila melanogaster, Caenorhabditis elegans, and Plasmodium falciparum, do not require Sec to reduce small molecule substrates, including 5,5'-dithiobis(2-nitrobenzoic acid), lipoic acid, selenite, and selenocystine. The data show 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 co-workers [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 access of the substrate to the N-terminal redox center and because they can make use of alternative mechanistic pathways that are not available to TR1.

Dataset Information

Synthesis of peptide substrates for mammalian thioredoxin reductase.

Publications

Synthesis of peptide substrates for mammalian thioredoxin reductase.

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