Cystine residues elimination

The first reaction is p-elimination in cysteine, serine, phosphoserine, and threonine residues due to attack by hydroxide ion, leading to the formation of very reactive dehydroalanine (DHA). In a cystine residue, this results in rupturing of the disulfide bond and liberation of a sulfide ion and free sulfur (Figure 13.4). Nucleophilic additions of the s-amino group of the protein-bound lysine to the double bond of DHA residue causes crosslinking of the polypeptide chain. After hydrolysis, a mixture of L-lysino-L-alanine and L-lysino-D-alanine, with probably a small proportion of dl and dd isomers,... 

Elimination reactions 526, 530, 677—690 beta, of cystine residues 85 conjugative 689 decarboxylative 689 facilitation by carbonyl group 681 of y substituent 746 of PLP-dependent enzymes 742 reversibility 690 Ellman s reagent 125,125s Elongation factor EF-Tu 558 Elongin complex 564... 

In a classic study on bovine pancreatic ribonuclease A at 90°C and pH conditions relevant for catalysis, irreversible deactivation behavior was found to be a function of pH (Zale, 1986) at pH 4, enzyme inactivation is caused mainly by hydrolysis of peptide bonds at aspartic acid residues as well as deamidation of asparagine and/or glutamine residues, whereas at pH 6-8, enzyme inactivation is caused mainly by thiol-disulfide interchange but also by fi-elimination of cystine residues, and deamidation of asparagine and/or glutamine residues. 

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 mechanism of disulfide reduction by phosphines is hypothesized to involve a stable intermediate containing a sulphur-phosphorous bond (6). Beta elimination would yield the phosphine sulfide and dehydroalanine. The formation of relatively stable adducts between cystine-containing peptides and the reagent was confirmed by mass spectrometry for several peptides with the major adduct representing one reagent molecule per cystine residue. 

Neither of the linkages formed by reactions (XXI) or (XXII) has been demonstrated in alkali-treated wool, but Patchornik and Sokolovsky (1964) and Bohak (1964) have demonstrated recently the presence of -iV-(D,L-2-amino-2 carboxymethyl)-L-lysine in hydrolyzates of proteins treated with alkali. This they attribute to the reaction of the -NH2 group of lysine residues with a-aminoacrylic acid residues formed from cystine residues by the 8-elimination reaction (Section V,A,5). Ziegler (1964) has shown that this amino acid residue is formed during the alkali treatment of wool in both the stretched and unstretched states. No assessment of the importance of these linkages in retaining set has yet been reported. 

Beta elimination high-temperature treatment of proteins leads to destmction of disulfide bonds as a result of yS-elimination from the cystine residue. 

The inactivation of proteins at high temperatures is often due to / -elimination of disulfides from the cystine residue, although other amino acids including Cys, Ser, Thr, Phe, and Lys can be degraded via / -elimination, as seen from Scheme 11.4. The inactivation is particularly rapid under alkaline conditions and is also influenced by the presence of metal ions. 

Although the results are consistent with a B-elimination reaction leading to formation of dehydroalanine, the conclusions are based on the assumption that the absorbance at 241 nm is associated with dehydroalanine side chains derived from cystine residues. This assumption may not always be justified for the following reasons. First, alkali treatment of casein which has very few or no disulfide bonds also yields significant amounts of dehydroalanine residues (52). These presumably arise from serine side chains. Second, Nashef et al. (41) cite evidence that other functionalities may contribute to the 241 nm absorption. These considerations suggest that there is a need to directly measure dehydroalanine in proteins. This is now possible with our method (52), whereby the dehydroalanine residues are first transformed to S-pyridylethyl side chains by reaction with 2-mercaptopyridine (Figure 12). Amino acid analysis of the acid-hydrolyzed protein permits estimation of the dehydroalanine content as S-B-(2-pyridylethyl)-L-cysteine along with the other amino acids. 

In the case of cystine, the eliminated thiolcysteine can form a second dehydroalanine residue ... 

Friedman et al 136) eliminate interference due to cysteine and cystine residues by reducing the protein disulfide bonds and alkylating the native and generated-SH groups with vinyl derivatives. Juneja et al. 219) evaluated in detail the interference by sulfur amino acids they estimated the tryptophan content of wool colorimetrically after hydrolysis with 6N Ba(OH)2 for 5 hours at 125° C followed by removal of liberated H2S and barium with silver sulfate and sulfuric acid respectively. 

Vithayathil et al. (34%). In 0.5 M HC1 at 30° RNase-A undergoes structural alterations which can be detected chromatographically at neutral pH. However, all the products are equally active enzymically, and no reaction would have been detected by assay. At pH 11.0 even more involved structural changes take place quite rapidly. Irreversible alkaline denaturation takes place at higher pH and is very rapid at 13. Here the activity loss is accompanied by marked spectral changes indicating reactions such as / elimination at cystine or serine residues (343). A temperature-induced isomerization at neutral pH has been reported by French and Hammes. This is discussed in a later section on nucleotide binding. 

Other conversions to unnatural residues occur when most proteins are exposed to high pH (80, 81,82). The high pH causes a -elimination of a cystine (see Figure 16) or O-substituted serine or threonine, with the formation of a dehydroalanine or a dehydro-a-aminobutyrate. Such products are subject to nucleophilic attack by the e-amino group of a lysine to form a cross-linkage, such as lysinoalanine, or attack by cysteine to form lanthionine. Walsh et al. (81) have taken advantage of the formation of these cross-links to produce avian ovomucoids that have nonreducible cross-links and have lost the antiprotease activity of one of their two inhibitory sites (see Figure 17). 

The generally accepted route of formation of LAL is through the formation of dehydroalanine from cysteine, cystine, serine or phosphoserine through e-elimination reaction followed by Michael addition between the dehydroalanine and the e-amino group of lysine. The formation of LAL from the oxidized derivatives of cystine has been reported by Finley et al. (13). It was suggested that oxidation of cystine to cystine monoxide may accelerate dehydroalanine formation and subsequent LAL formation. It was also observed that very little LAL was formed through the 6-elimination of cysteine. Mel let (14) proposed that the elimination reaction in serine residues was responsible for the formation of dehydroalanine in peptides. Whitaker and Feeney (15) have reviewed the alkaline decomposition of phosphoserine and glycosylated serine or threonine residues in proteins. 

Cyanide-nutrient interactions are reported for alanine, which appears to exacerbate cyanide toxicity, and for cystine, which seems to alleviate toxicity. Dietary cyanide - at levels that do not cause growth depression -alleviates selenium toxicity in chickens, but not the reverse. For example, dietary selenium, as selenite, at lO.Omg/kg for 24 days, reduced growth, food intake, and food utilization efficiency, and produced increased liver size and elevated selenium residues the addition of 45.0 mg CN/kg diet (100.0 mg sodium nitroprusside/kg) eliminated all effects except elevated selenium residues in liver. The mechanism of alleviation is unknown and may involve a reduction of tissue selenium through selenocyanate formation, or increased elimination of excess selenium by increasing the amount of dimethyl selenide exhaled. At dietary levels of 135.0 mg CN/kg plus 10.0 mg selenium/kg chick growth was significantly decreased. This interaction can be lost if there is a deficiency of certain micronutrients or an excess of vitamin K. 

A postulated mechanism for lysinoalanine formation is a two-step process. First, hydroxide ion-catalyzed elimination reactions of serine, threonine, and cystine give rise to a dehydroalanine intermediate, illustrated in Figures 9 and 10 for cystine. The dehydroalanine residue, which contains a conjugated carbon-carbon double bond, then reacts with the e-NH2 group of lysine to form a lysinoalanine crosslink. 

The cited evidence for the B-elimination mechanism leading to dehydroalanine formation merits further comment. Nashef et al. (41) report that alkali-treatment of lysozyme ribonuclease and several other proteins resulted in loss of cystine and lysine residues and the appearance of new amino acids lysinoalanine, lanthionine, and B-aminoalanine. Alkali-treatment of the proteins induced an increase in absorbance at 241 nm, presumably from the formation of dehydroalanine residues. The dehydroalanine side chain can participate in nucleophilic addition reactions with the e-NH2 group of lysine to form lysinoalanine, with the SH groups of cysteine to form lanthionine, and with ammonia to form B-aminoalanine. 

Effect of sulfur amino acids. Lysinoalanine and related cross-1 inked amino acids may be derived from reaction of lysine with dehydroalanine residues formed by elimination reactions from serine, cystine, and possibly cysteine residues in proteins. Threonine residues can, in principle, react similarly to form methylated homo-logues (Friedman, 1977). The double bond of dehydroalanine, which... 

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