Disulfide groups, thioredoxin reductase

Mammalian thioredoxin reductases are a family of selenium-containing pyridine nucleotide-disulfide oxidoreductases. These enzymes catalyze NADPH-dependent reduction of the redox protein thioredoxin (Trx), which contains a redox-active disulfide and dithiol group and by itself may function as an efficient cytosolic antioxidant [77]. One of the functions of Trx/ thioredoxin reductase system is the NADPH-catalyzed reduction of protein disulfide [78] ... 

In the first step, thioredoxin reductase reduces a small redox protein, thioredoxin, via enzyme-bound FAD. This involves cleavage of a disulfide bond in thioredoxin. The resulting SH groups in turn reduce a catalytically active disulfide bond in nucleoside diphosphate reductase ( ribonucleotide reductase ). The free SH groups formed in this way are the actual electron donors for the reduction of ribonucleotide diphosphates. 

The reaction catalyzed by the first of these is illustrated in Table 15-2 (reaction type F). The other two enzymes usually promote the reverse type of reaction, the reduction of a disulfide to two SH groups by NADPH (Eq. 15-22). Glutathione reductase splits its substrate into two halves while reduction of the small 12-kDa protein thioredoxin (Box 15-C) simply opens a loop in its peptide chain. The reduction of lipoic acid opens the small disulfide-containing 5-membered ring in that molecule. Each of these flavoproteins also contains within its structure a reducible disulfide group that participates in catalysis. 

In neural cells, the redox status is controlled by the thioredoxin (Trx) and glutathione (GSH) systems that scavenge harmful intracellular ROS. Thioredoxins are antioxidants that serve as a general protein disulphide oxidoreductase (Saitoh et al., 1998). They interact with a broad range of proteins by a redox mechanism based on the reversible oxidation of 2 cysteine thiol groups to a disulphide, accompanied by the transfer of 2 electrons and 2 protons. These proteins maintain their reduced state through the thioredoxin system, which consists of NADPH, thioredoxin reductase (TR), and thioredoxin (Trx) (Williams, Jr. et al., 2000 Saitoh et al., 1998). The thioredoxin system is a system inducible by oxidative stress that reduces the disulfide bond in proteins (Fig. 7.4). It is a major cellular redox system that maintains cysteine residues in the reduced state in numerous proteins. 

The pyridine nucleotide-disulfide oxidoreductases, lipoamide dehydrogenase (4), glutathione reductase (5), and thioredoxin reductase (6-8) share so many properties in common that they will be compared and contrasted before being considered separately. As their group name implies, they catalyze the transfer of electrons between pyridine nucleotides and disulfides. In spite of their similarities they function in widely divergent metabolic roles. 

Glutathione reductase, thioredoxin reductase, and lipoamide dehydrogenase are members of a group of flavoproteins that contain an active site disulfide as well as FAD. They catalyze the NAD(P)H dependent reduction of a disulfide... 

The actual process, which is catalyzed by ribonucleotide reductase, is more complex than the preceding equation would indicate and involves some intermediate electron carriers. The ribonucleotide reductase system from E. coli has been extensively studied, and its mode of action gives some clues to the nature of the process. Two other proteins are required, thioredoxin and thioredoxin reductase. Thioredoxin contains a disulfide (S—S) group in its oxidized form and two sulfhydryl (—SH) groups in its reduced form. NADPH reduces thioredoxin in a reaction catalyzed by thioredoxin reductase. The reduced thioredoxin in turn reduces a ribonucleoside diphosphate (NDP) to a deoxyribonucleoside diphosphate (dNDP), shown in Figure 23.31b, and this reaction is actually catalyzed by ribonucleotide reductase. Note that this reaction produces dADP, dGDP, dCDP, and dUDP. The first three are phosphorylated to... 

The E. coli enzyme has been purified to homogeneity the molecular weight is about 66,000 and the enzyme consists of two identical or very similar polypeptide chains. The amino acid composition of the enzyme has been determined (13). It has been proposed that each of the two thioredoxin reductase subunits has an active center containing one FAD molecule and one disulfide linkage, both of which act as oxidation-reduction acceptors during catalysis (13). As well, thioredoxin reductase contains four additional sulfhydiyl groups per molecule (presumably, two per subunit), but these do not participate in the catalytic function. The amino acid sequence at the active site of the E. coii thioredoxin reductase has been determined to be as follows (14) ... 

Regeneration of reduced enzyme In order for ribonucleotide reductase to continue to produce deoxyribonucleotides, the disulfide bond created during the production of the 2 -deoxy carbon must be reduced. The source of the reducing equivalents is thioredoxin—a peptide coenzyme of ribonucleotide reductase. Thioredoxin contains two cysteine residues separated by two amino acids in the peptide chain. The two sulfhydryl groups of thioredoxin donate their hydrogen atoms to ribonucleotide reductase, in the process forming a disulfide bond (see p. 19). 

Ribonucleotides are reduced to the 2 -deoxyribo-nucleotides (Eq. 16-21) that are needed for DNA synthesis by enzymes that act on either the di- or triphosphates of the purine and pyrimidine nucleosides348-351 (Chapter 25). These ribonucleotide reductases utilize either thioredoxin or glutaredoxin (Box 15-C) as the immediate hydrogen donors (Eq. 16-22). The pair of closely spaced -SH groups in the reduced thioredoxin or glutaredoxin are converted into a disulfide bridge at the same time that the 2 -OH of the ribonucleotide di- (or tri-) phosphate is converted to H20. While some organisms employ a vitamin B12-... 

TrxRs are homodimeric flavoproteins [80] that catalyze the NADPH-dependent reduction of thioredoxin (Trx), a ubiquitous 12 kDa protein that is the major protein disulfide reductase in cells [81], and belongs to the pyridine nucleotide-disulfide oxidoreductase family [82]. Each monomer includes an FAD prosthetic group, a NADPH binding site and an active site containing a redox-active selenol group. Electrons are transferred from NADPH via FAD to the active-site selenol of TrxR, which then reduces the substrate Trx [83]. The crystal structure of TrxR is shown in Fig. 13 [84],... 

The deoxynucleotides, that is, the nucleotides where the hydroxyl at C2 of the ribose unit is missing (including thymidine phosphate), are generated from the nucleotides where the hydroxyl group is present using a thioredoxin disulfide on the diphosphates (ribonucleoside diphosphate reductase, EC 1.17.4.1). In this way, ribonucleo-... 

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