Thioredoxin reductase cystine residues

Electron transfer between pyridine nucleotides and disulfide compounds is catalyzed by several fiavoproteins and three of these are well characterized. Lipoamide dehydrogenase functions in the oxidative decarboxylation of a-keto acids catalyzing the reoxidation of reduced lipoate by NAD+ (18, 19). Glutathione reductase catalyzes electron transfer between NADPH and glutathione ZO-22). Thioredoxin reductase catalyzes the reduction of thioredoxin by NADPH (5) thioredoxin is a protein of 12,000 molecular weight containing a single cystine residue which is the electron acceptor S3). 

It is not surprising that enzymes catalyzing such similar chemical reactions should bear striking similarity to one another both structurally and mechanistically. Lipoamide dehydrogenase (34-38), glutathione reductase (39), and thioredoxin reductase (SO, 31) contain, in addition to FAD, a reactive disulfide which is functional in catalysis. These fiavoproteins consist of two identical or near identical polypeptide chains, each with a reactive cystine residue, and two molecules of FAD (31-36). 

Regeneration of the ribonucleotide reductase is accomplished in Escherichia coli and in mammals by thioredoxin, a dithiol polypeptide (M.W. 12,000) coenzyme, which also plays a role in other protein disulfide reductase reactions. In thioredoxin, two cysteine residues in the sequence -Cys-Gly-Pro-Cys are converted to cystine. Reduced thioredoxin is regenerated by thioredoxin reductase, a flavoprotein enzyme that uses NADPH + H+. 

Thus thioredoxin reductase, like its substrate thioredoxin, contains only two amino acid residues between the two half cystine residues and in both proteins the disulfide defines a small loop of only 14 atoms. 

Thioredoxin reductases have also been purified from yeast (43, 134) and from rat fiver (135). Thioredoxin reductase from yeast is a flavo-protein with a molecular weight of approximately 75,000 and consists of two subunits, each containing one molecule of FAD. Although the amino acid composition of this thioredoxin reductase is quite different from that of E. coli, both enzymes contain 5 half-cystine residues and have almost identical absorption spectra. Like the E. coli enzyme, thioredoxin reductase from yeast is completely inhibited by p-chloro-mercuriphenylsulfonate (PCMS) only in the presence of NADPH suggesting that the yeast enzyme also contains a disulfide bridge at the catalytic site. 

Thioredoxin (1,2) are small (M. 11-12,000) ubiquitous redox proteins with two half-cystine residues in the conserved active site structure Trp-Cys-Gly-Pro-Cys. The oxidized form Trx-S2 is reduced by NADPH and thioredoxin-reductase the reduced rorm Trx(SH)2 is a powerful protein disulfide oxido-reductase which regulatesthe activity of enzymes by thiol redox control it serves as hydrogen donor for various reductive enzymes such as ribonucleotide reductase or enzymes reducing sulfate or methionine sulfoxide. Also, Trx(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and assembly of filamentous phages (fl and M13), at least in E, ooti. 

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