Thioredoxin active site

Dillet V, Dyson HJ, Bashford D (1998) Calculations of electrostatic interactions and pKas in the active site of Escherichia coli thioredoxin. Biochemistry 37 10298-10306. 

Deoxynucleotides for DNA synthesis are made at the nucleoside diphosphate level and then have to be phosphorylated up to the triphosphate using a kinase and ATP. The reducing equivalents for the reaction come from a small protein, thioredoxin, that contains an active site with two cysteine residues. Upon reduction of the ribose to the 2 -deoxyri-bose, the thioredoxin is oxidized to the disulfide. The thioredoxin(SS) made during the reaction is recycled by reduction with NADPH by the enzyme thioredoxin reductase. 

A likely mechanism for the ribonucleotide reductase reaction is illustrated in Figure 22-41. The 3 -ribonu-cleotide radical formed in step (T) helps stabilize the cation formed at the 2 carbon after the loss of H20 (steps and (3)). Two one-electron transfers accompanied by oxidation of the dithiol reduce the radical cation (step ). Step (5) is the reverse of step ( ) regenerating the active site radical (ultimately, the tyrosyl radical) and forming the deoxy product. The oxidized dithiol is reduced to complete the cycle (step ). Ini , coli, likely sources of the required reducing equivalents for this reaction are thioredoxin and glutaredoxin, as noted above. 

If there are three or more -SH groups in a chain some incorrect pairing may, and often does, occur. Tire protein disulfide isomerases break these bonds and allow new ones to form.92 The active sites of these isomerases contain pairs of -SH groups which can be oxidized to internal -S-S- bridges by NAD+-dependent enzymes. These enzymes and their relatives thioredoxin and glutaredoxin are discussed further in Box 15-C. Glutathione and oxidation-reduction buffering are considered in Box 11-B. 

It was a surprise to discover that a mutant of E. coli lacking thioredoxin can still reduce ribonucleotides. In the mutant cells thioredoxin is replaced by glutaredoxin, whose active site disulfide linkage is reduced by glutathione rather than directly by NADPH. Oxidized glutathione is, in turn, reduced by NADPH and glutathione reductase. Thus, the end result is the same with respect to ribonucleotide reduction. 

A variation is observed for E. coli thioredoxin reductase. The reducible disulfide and the NADPH binding site are both on the same side of the flavin rather than on opposite sides as in Fig. 15-12.190/259 Mercuric reductase also uses NADPH as the reductant transferring the 4S hydrogen. The Hg2+ presumably binds to a sulfur atom of the reduced disulfide loop and there undergoes reduction. The observed geometry of the active site is correct for this mechanism. 

A reversible covalent modification that plants use extensively is the reduction of cystine disulfide bridges to sulf-hydryls. Many of the enzymes of photosynthetic carbohydrate synthesis are activated in this way (table 9.3). Some of the enzymes of carbohydrate breakdown are inactivated by the same mechanism. The reductant is a small protein called thioredoxin, which undergoes a complementary oxidation of cysteine residues to cystine (fig. 9.5). Thioredoxin itself is reduced by electron-transfer reactions driven by sunlight, which serves as a signal to switch carbohydrate metabolism from carbohydrate breakdown to synthesis. In one of the regulated enzymes, phosphoribulokinase, one of the freed cysteines probably forms part of the catalytic active site. In nicotinamide-adenine dinucleotide phosphate (NADP)-malate dehydrogenase and fructose-1,6-bis-... 

Phylogenetic data from 18 sequences of the thioredoxin family were used to calculate the eloping scheme for active site positions [13]. The frequency of different amino acids at a given position determines a positional mixture with different... 

Fig. 7.4 Reactions associated with the thioredoxin system. Thioredoxin is a redox-regulating protein with a redox-active disulfide/dithiol within the conserved active site sequence -Cys-Gly-pro-Cys-. Thioredoxin reductase, a 55 kDa flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (1) and thioredoxin oxidase (2), a flavin-dependent sulfhydry 1 oxidase that catalyzes the oxidative protein folding with the generation of disulfides...

A number of biochemical reactions involve proteins as reactants, and so it is important to be able to determine the standard transformed Gibbs energies of formation of their reactive sites at specified pH. The standard transformed Gibbs energies of formation of the active sites of ferredoxin, cytochrome c, and thioredoxin are given in tables discussed earlier in Chapter 4. 

Holmgren, A. 1995. Thioredoxin structure and mechanism Conformational changes on oxidation of the active-site sulfhydryls to a disulfide. Structure 3 239-243. 

Protein structure determinations have identified several examples of one domain inserted within another. One example is the E. coli DsbA protein, which catalyzes the formation of disulfide bonds in the periplasm. The enzyme consists of two domains a thioredoxin-like domain that contains the active site, and an inserted helical domain similar to the C-terminal domain of thermolysins (Martin et al., 1993). The inserted domain forms a cap over the active site, suggesting that it plays a role in binding to partially folded polypeptide chains before oxidation of... 

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 discovery of non-specific disulfide reductases which are labile in aerobic cellular extracts suggests that kinetic constraints of thiol/disulfide exchange in vivo are very complex. One of such proteins is thioredoxin which behaves as a non-specific protein-disulfide reductase. Thioredoxin also works as a cofactor of sulfoxide reductases. The dithiol active site of thioredoxin sits on a protrusion of the protein surface [274], Thioredoxin is an ubiquitous protein whose molecular weight is about 12 KDa [274,275], It has been found in cytosolic and mitochondrial [276] compartments of animal cells, and it is partly bound to membranes. High contents in thioredoxin have been found in neurons, secretory and epithelial cells. Redox recycling of thioredoxin is insured by thioredoxin reductase, which has been identified in a variety of mammalian cells as a symmetrical dimer with a molecular weight of 116KDa[274]. Thioredoxin reductase is NADPH-specific, but it exhibits a very wide disulfide substrate specificity. 

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... 

FIGURE 2. Three-dimensional structure of conserved residues and the substrate GDP in the active site of E. coli protein R1 (A) (Eriksson et al., 1997), and proposed reaction mechanism for RNR (B), A turnover is initiated by long-range RTF between Tyrl22 in R2 and Cys439 in Rl, and completed by a reversal of this RTP (thin arrows). Oxidised Rl is reduced by the thioredoxin (Trx), or glutaredoxin system. 

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