Cysteine disulfide interchange

FIGURE 6.22 Disulfide interchange.92 (A) Discovered in synthesis when hydrazinolysis of an unsymmetrical derivative of cystine gave two symmetrical products instead of the expected monohydrazide at the urethane-protected cysteine moiety of the derivative.95 (B) Mechanism for interchange catalyzed by strong acid,94 which is suppressed by thiols. (C) Mechanism for interchange catalyzed by weak alkali, which is enhanced by thiols. 

Further structure-activity relationship (S AR) analyses of other cytoprotective enzyme inducers revealed the fact that all inducers can react with thiol/disulfide groups by alkylation, oxidoreduction, or thiol-disulfide interchange [Dinkova-Kostova and Talalay, 1999]. In fact, the capability of enzyme inducers to induce cytoprotective enzymes is well correlated with their reactivity with thiols. These results suggested a cellular sensor of inducers with highly reactive sulfhydryl groups, possibly reactive thiols in cysteine residues of a sensor protein. Nevertheless, the initial search for the sensor protein by using radioactively labeled inducers was not successful due to the abundance of thiol groups presented in many proteins in cells [Holtzclaw et al., 2004]. The molecular mechanism by which cytoprotective enzymes are induced remained to be elucidated. 

To ascertain the upper limit of protein thermostability and to evaluate the effect of additional disulfide bridges on the enhancement of protein thermostability, additional cysteine residues were introduced into several unrelated proteins by site-directed mutagenesis and deactivation behavior tested at 100°C (Volkin, 1987). All the proteins investigated underwent heat-induced beta-elimination of cystine residues in the pH 4—8 range with first-order kinetics and similar deactivation constants kj that just depended on pH 0.8 0.3 h-1 at pH 8.0 and 0.06 0.02 h 1 at pH 6.0. These results indicate that beta-elimination is independent of both primary amino acid sequence and the presence of secondary structure elements. Elimination of disulfides produces free thiols that cause yet another deleterious reaction in proteins, heat-induced disulfide interchange, which can be much faster than beta-elimination. 

The cystine residues, because they may act as cross-linkages between protein chains, have been studied more closely than other residues in keratin. Burley s (1956a) use of the concept of thiol-disulfide interchange to explain the effects of chemical modification on the physical properties of wool fibers has stimulated further work on cysteine and cystine residues. 

Zahn et al (1960) have suggested that lanthionine may be an inevitable side product of thiol-disulfide interchange reactions involving cysteine and... 

There are at least two types of enzyme systems involved in the formation and breakage of disulfide bonds of cystine residues in proteins. A thiol-disulfide interchange enzyme (protein disulfide-isomerase EC 5.3.4.1 other name, S-S-rearrangase) was first described in 1963 ( 47) and was subsequently purified from beef liver (48,49). The molecular weight of the enzyme is 42,000. The enzyme contains three half-cystine residues, one of which must be cysteine in order for the enzyme to be active. The enzyme catalyzed the rearrangement of random incorrect pairs of half-cystine residues to the native disulfide bonds in several protein substrates. Low levels of mercaptoethanol were required for activity unless the enzyme was reduced prior to use. The efficiency of the enzyme in catalyzing the interconversion of disulfide bonds was found to be a function of the number of disulfide bonds in the substrate. Purification of a thiol-disulfide interchange enzyme from Candida claussenii has been described recently (50). 

It has also been possible to modulate enzyme function (29) by introduction of a disulfide bridge spanning the active site cleft of T4 lysozyme (Fig. 1). In order to avoid possible thiol-disulfide interchange with Cys-S4 and Cys-97 in the native structure, these two residues were converted to threonine and alanine, respectively, with no loss in the activity or stability of the enzyme. The latter protein was then further modified by replacing Thr-21 and Thr-42 by cysteines that spontaneously oxidized to the desired disulfide. This oxidized enzyme form exhibited no detectable activity, although some activity (7% of wild type) was restored on reduction of the linkage. This represents a novel use of the disulfide bond to modulate catalytic activity. 

The reactions of cystine with base are more complicated and not completely elucidated. Among products identified are cysteine, cysteinesulfinic acid, 5-sulfocysteine, cysteic acid, acetic acid, thiosulfate, and the persulfide H02CCH(NH2)CH2-S-SH (DeMarcoeta/., 1%3 Danehy and Hunter, 1%7). Disulfide interchange (see Section II1,C) can also take place in base (Ryle and Sanger, 1955). 

Most inhibitors of chymotrypsin and trypsin contain disulfide bonds which are needed to maintain active conformations and configurations. Since thiols are expected to react with inhibitor disulfide bonds via sulfhydryl-disulfide interchange and oxidation-reduction reactions, illustrated in Figure 1, we carried out extensive studies on the ability of cysteine, N-acetylcysteine, and reduced glutathione to synergize heat inactivation of soybean inhibitors. Figures 2 and 3 depict some of our findings. 

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. 

Cysteamine has been shown to be effective in removing the lysosomal cystine in cystinosis. The weak base cysteamine tends to distribute within the acidic lysosomal space. A mixed disulfide of cysteamine and cysteine is formed by disulfide interchange and is transported out of the lysosomal space by the carrier for lysine. In cytosol the mixed disulfide is cleaved by reduced glutathione. Early introduction of cysteamine therapy can protect the kidneys from further progression of glomerular destruction. In end-stage renal failure replacement therapy (dialysis, transplantation) becomes necessary. Longterm survival of cystinotic patients is followed by additional late sequelae e.g. distal myopathy, loss of retinal function (blindness), disturbances of memory and other cerebral functions (for review see [3]). 

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