Active disulfide

The thioredoxin domain (see Figure 2.7) has a central (3 sheet surrounded by a helices. The active part of the molecule is a Pa(3 unit comprising p strands 2 and 3 joined by a helix 2. The redox-active disulfide bridge is at the amino end of this a helix and is formed by a Cys-X-X-Cys motif where X is any residue in DsbA, in thioredoxin, and in other members of this family of redox-active proteins. The a-helical domain of DsbA is positioned so that this disulfide bridge is at the center of a relatively extensive hydrophobic protein surface. Since disulfide bonds in proteins are usually buried in a hydrophobic environment, this hydrophobic surface in DsbA could provide an interaction area for exposed hydrophobic patches on partially folded protein substrates. 

Fox B, CT Walsh (1982) Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction active disulfide. J Biol Chem 257 2498-2503. 

As outlined in previous sections, escape of polyplexes from endosomes to the cytosol can be a major bottleneck in delivery. Membrane-active polymer domains or other conjugated molecules can help to overcome this barrier (see Sect. 2.3), but they may trigger cytotoxicity when acting extracellularly or at the cell surface. Therefore membrane-crossing agents either have to be inherently specific for endo-somal compartments (for example by pH-specificity), or they have to be modified to be activated in endosomes. For example, the reducing stimulus of intracellular vesicles has been used to activate formulations containing less active disulfide precursors of LLO [163] or Mel [170]. 

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] ... 

The third enzyme, dihydrolipoamide dehydrogenase [E3], reoxidizes dihydrolipoamide, with NADH+H"" being formed. The electrons are first taken over by enzyme-bound FAD (3a) and then transferred via a catalytically active disulfide bond in the E3 subunit (not shown) to soluble NAD"" (3b). 

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. 

This EAD-dependent enzyme [EC 1.6.4.2] catalyzes the reaction of NADPH with glutathione disulfide to produce NADP+ and two glutathione molecules. The enzyme activity is dependent on a redox-active disulfide group in each of the active sites. 

A tryptic fragment with three interchain disulfide bonds between four peptide chains was synthesized by repetitive application of this Cys activation/disulfide forming method, and by this procedure the disulfide connectivities of synthetic porcine C5a-anaphylatoxin was confirmed to be identical to those for the natural peptide. 67 ... 

Thiols can be linked to insoluble supports as disulfides by disulfide interchange. Mixed disulfides can be prepared on insoluble supports by treating support-bound thiols with excess activated disulfide (e.g. 2-benzothiazolyl, 2-nitrophenyl, 3-nitro-2-pyridyl disulfides [60,676] or a methanethiosulfonate MeS02-SR [677] Figure 3.33), or by treating a support-bound disulfide (e.g. a 2-pyridyl disulfide [191]) with a thiol. Resin-bound disulfides are stable under the conditions of standard Fmoc peptide synthesis, but can be cleaved by reducing agents (Entries 8 and 9, Table 3.37 [191,676,678-681]). 

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...

Both MAO-A and MAO-B contain a redox-active disulfide at the catalytic center. The results imply that MAO may be a novel type of disulfide oxidoreductase and may open the way to characterizing the catalytic and chemical mechanism of the enzyme. 

Thelander, L., 1974, Reaction mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Oxidation-reduction-active disulfides in the B1 subunit. J. Biol. Chem. 249 4858n4862. 

Thioredoxin reductase (TrxR) acts in the reverse direction and shows a somewhat different mechanism, which is dependent on the protein source. Prokaryotes, plants, and lower eukaryotes contain a 35-kDa TrxR with one redox-active disulfide. Higher eukaryotes produce a 55-kDa TrxR that has either an additional redox-active disulfide or a selenenylsulfide in the flexible C-terminal part of the neighboring subunit (15). In low Mr TrxR, a large conformational change is required to move reducing equivalents from the apolar flavin site to the surface of the protein where the thioredoxin redox partner binds. In high Mr TrxR, this transfer is mediated by the second disulfide or selenylsulfide, and the conformational changes required are comparatively small (17). 

Thioredoxin from E. coli has been studied extensively using biochemical, spectroscopic and X-ray diffraction techniques. The protein consists of a single polypeptide chain of 108 amino acid residues of known sequence. The protein has been cloned and expressed. Thioredoxin of E. coli is a compact molecule with 90% of its residues in hehces, beta-strands or reverse turns. This protein transports electrons via an oxidation-reduction active disulfide". The oxidized form thioredoxin-(S2) is reduced to thioredoxin-(SH)2. In particular, this protein was found to participate in the reduction of ribonucleotides to deoxyribonucleotides. In Fig. 1, the optimized stracture is shown with a carbon backbone for clarity only. The molecule consists of two conformational domains, connected by two helices. The beta-sheet forms the core of the molecule packed on either side by clusters of hydrophobic residues. Helices form the external surface. We used a crystal stracture of the oxidized form of thioredoxin from Escherichia coli that has been refined by the stereochemically restrained least-squares procedure at 1.68 A resolution". ... 

FDA (Center for Drug Evaluation and Research). 1999. List of Drug and Pood that Contain Intentionally Introduced Mercury Compounds. Updated November 17, 1999. Online. Available http //www.fda.gov/cder/fdama/mercury300.htm Fox, B., and C.T Walsh. 1982. Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide. J. Biol. Chem. 257(5) 2498-2503. 

In the broadest terms, cell-free synthesis offers advantages over traditional in vivo methods of recombinant protein expression, as it enables the experimentalist strictly to control conditions under which expression reactions are performed. Benefits of such an open expression system are exemplified by the fact that cell-free reaction conditions can be sufficiently modified to support co-translational folding of active disulfide-bonded proteins. 

In proteins that contain more than one disulfide bond, the proper pairing of cysteine residues Is essential for normal structure and activity. Disulfide bonds commonly are... 

Mercuric ion reductase, the FAD-containing merA gene product, has several pairs of conserved cysteines. From site-specific mutagenesis studies, cysteine residues in the sequence 134-Thr-Cys-Val-Asn-Val-Gly-Cys-140 are known to comprise a redox-active disulfide group in addition, a redox-inactive pair of cysteines near the carboxyl terminus is also required for the selective reduction of Hg(II). Exactly how the enzyme achieves the chemistry shown in Equation... 

Much less is known about the final step of the pathway, specifically the Bi2-dependent conversion of o( into queuosine. In his review of queuosine biosynthesis, Iwata-Reuyl draws a parallel between the reduction of o( and the reaction performed by ribonucleotide reductases and proposes a mechanism involving a thiyl radical and redox-active disulfide (Figure 36). 

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