Oxidation of cysteine residues

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-... 

Phenol nitration with tetranitromethane (37) (TNM) is typically run at pH 8 at room temperature and is selective for tyrosine residues under these conditions (although some oxidation of cysteine residues has been reported) (38). The product of this reaction could be thought to develop through an electrophilic... 

Auto-oxidation of cysteine residues during cleavage of the disulfide bridge-containing proteins is a potential concern. This concern can be addressed by first reducing those proteins at alkaline pH ( 8.0) with either 2-mercaptoethanol or dithiothreitol (Equation (1)) and then alkylating with iodoacetic acid to S-carboxymethyl derivatives (Equation (2)) The reduction-alkylation process also disrupts the 3D structure of proteins to allow more sites accessible for cleavage. 

Reactivity is not limited to direct addition to the phenoxyl ring structure, and reactions with thiols, amines and olefmic compounds have been reported. Both HOBr and HOCl can elicit oxidation of cysteine residues, where oxidation of the thiol moiety to a sulphinic acid is associated with activation of the latent form of matrix metalloproteinase-7 [111]. Higher doses of HOCl cause inactivation of the enzyme through site-specific modification of tryptophan and the adjacent glycine to yield an unknown product that lacks four mass units [112]. 

Glutathionylation may inhibit proteins, yet there is an important difference from inactivation by oxidation of cysteine residues by ROS, forming sulfinic or sulfonic acids. Both cysteine oxidation states are generally regarded as not reducible under cellular conditions although the cellular reduction and reactivation of a peroxiredoxin cysteine sulfinic acid has recently been described ... 

The reversible oxidation of cysteine residues has been shown to function as a switch between different states of activity of transcription factors. This has been shown, e.g., for the transcription factor API, which contains cysteine motifs that regulate activity in response to oxidative stress (Karimpour et al., 2002). 

Polypeptides are polymers composed of amino acids linked by peptide bonds. The order of the amino acids in a polypeptide is called the amino acid sequence. Disulfide bridges, formed by the oxidation of cysteine residues, are an important structural element in polypeptides and proteins. 

Scheme 82. Formation of a disulfide bond through oxidation of cysteine residues.

Metabolic oxidation reactions may occur to the side chains of sulfor-containing residues, similar to that observed for in vitro chemical instability. Methionine can be oxidized to the sulfoxide, whereas metabolic oxidation of cysteine residues forms a disulfide. Metabolic reductive cleavage of disulfide bridges in proteins may occur, yielding free sulfhydryl groups. 

Disulphide bridges, produced by oxidation of cysteine residues. They are especially relevant in the stabilization of the three-dimensional structure of low molecular weight extracellular proteins. 

A later paper details the de novo design of peptides that switch between a coiled-coil and a helical-hairpin conformation. The parent peptide (coiled-coil switch peptide) CSP-1 is a parallel dimeric coiled coil, but oxidation of cysteine residues in (he peptide causes the switch to a monomer. It remains ar-helical, although the a-helical content is low. Increasing the loop length gives CSP-3, which is a peptide with a higher helical content. CSP-6, an anagram of CSP-3, is more helical stUl and can be switched between the helical-hairpin and coiled-coil conformations. 

Ironically, while water is critical in maintaining molecular shape, the aqueous state is not one in which proteins are long resistant to denaturation. A variety of environmental changes such as temperature, pH, salts and solvents can cause protein inactivation in the aqueous state, and the mechanisms of irreversible protein inactivation often follow conunon pathways. These include cystein destruction, thiol-catalyzed disulfide interchange, oxidation of cystein residues, deamidation of asparagine and glutamine residues and hydrolysis of peptide bonds at aspartic acid residues. 

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