Dehydroalanine residue

FIGURE 3.10 Deprotection of functional groups by beta-elimination.17 (A) Removal of a labile proton beta to a good leaving group leads to release of the protector as the didehydro compound. (B) Recently developed protectors (Samukov et al., 1988) also designated untra-ditionally as 4-nitrophenyl- (C) Transformation of an O-protected serine residue into a dehydroalanine residue by hefa-elimination. 

Fig. 25-15). In every case it is NH3 or an amine, rather than an OH group, that is eliminated. However, the mechanisms probably resemble that of fumarate hydratase. Sequence analysis indicated that all of these enzymes belong to a single fumarase-aspartase family.64 65 The three-dimensional structure of aspartate ammonia-lyase resembles that of fumarate hydratase, but the catalytic site lacks the essential HI 88 of fumarate hydratase. However, the pKa values deduced from the pH dependence of Vmax are similar to those for fumarase.64 3-Methylaspartate lyase catalyzes the same kind of reaction to produce ammonia plus czs-mesaconate.63 Its sequence is not related to that of fumarase and it may contain a dehydroalanine residue (Chapter 14).66... 

Dehydroalanine 755, 757s Dehydroalanine residue 754, 756 Dehydroascorbate 787 transport 416... 

The coupling reaction by which the aromatic group from one residue of mono- or diiodotyrosine is joined in ether linkage with a second residue is also catalyzed readily by peroxidases. One dehydroalanine residue is formed for each molecule of hormone released.108 A possible mechanism involves formation of an electron-deficient radical, which can undergo (3 elimination to produce a dehydroalanine residue and an aromatic radical. The latter could couple with a second radical to form triiodothyronine or thyroxine. However, as depicted in Eq. 25-6, the radical coupling may occur prior to chain cleavage. While P elimination (pathway... 

Figure 1. New amino acids which may be formed through reaction of a dehydroalanine residue with internal or external nucleophiles in alkali treated proteins.

Generally sulfonyl esters of serine are relatively stable with respect to reactivation by a nucleophile such as hydroxylamine (Alexander et al. 1963). However, treatment of the sulfonyl derivatives of chymotrypsin with 0.1 N NaOH at 0°C yields a dehydroalanine residue in... 

At a given temperature the overall rate of reaction depends on the rate of 13-elimination and on the conformation of the protein. This is because the accessibility of the dehydroalanine residue for the nucleophilic attack depends on the spatial arrangement of the reacting groups. The reaction can be inhibited by acylating the nucleophilic groups in proteins or by adding thiol compounds, which compete with a.a. residues for the dehydroalanine double bond ... 

Partial removal of the phosphate groups of phosvitin by -elimination in alkaline solution results in a decreased in vitro initial rate of hydrolysis by trypsin (268). The decreased rate of hydrolysis might be a result of (a) a change in conformation of the protein on removal of the phosphate groups, (b) cross-linking by the reaction of the dehydroalanine residues with lysine residues (to form lysinoalanine), or (c) racemization of some of the residues by the alkaline treatment. 

Theonella sp. collected off Hachijo-jima Island [30]. Theonellamide F (35) is a bicyclic peptide containing twelve aminoacids. In this cyclic peptide a dehydroalanine residue is masked by intramolecular Michael addition, giving rise to the elaborate and unprecedented histidino-alanine bridge. Further separation of the antifungal fraction of the sponge extract afforded five related peptides, theonellamides A-E [31]. Theonellamide A... 

This enz)one utilizes a divalent cation 40) and also an electrophilic group, probably an activated dehydroalanine residue 41) to catalyze the elimination of ammonia from histidine to form urocanate. 

The reactivity of the methyl ester also more closely emulates the reactivity of dehydroalanine residues in peptides. Gross et al. (22) observed that dehydroalanine was present in the antibiotics cinnaniycin and duramycin. Careful measurement of dehydroalanine in alkaline treated proteins was conducted by Sen et al. (17) and Walsh et al. (23) by change in absorbance as described by Carter and Greenstein (24). The measurements were difficult in pure proteins and would not seem to lend themselves to measurement of dehydroalanine in complex food protein systems. 

The formation of lanthionine in keratin libers is believed to involve two reaction sequences. The first sequence consists of beta-elimination to form dehydroalanine residues in hair ... 

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. 

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. 

Figure 10. Mechanism for base-catalyzed formation of one dehydroalanine residue, and one persulfide ion from a protein disulfide bond. The persulfide can decompose to a thiolate anion and elemental sulfur.

Figure 11. Mechanistic pathways for trapping dehydroalanine residues by RS and SO3-2, thus preventing lysinoalanine formation.

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... 

Thus, depending on the pH of the reaction, sulfhydryl groups of cysteine react about 34 to 5000 times faster than the e-amino groups of lysine with vinyl compounds such as N-acetyl dehydroalanine methyl ester (Friedman and Wall, 1964 Friedman et al., 1965 Cavins and Friedman, 1967 Snow et al., 1976). Therefore, by adding thiols such as cysteine it may be possible to prevent the formation of dehydroalanine residues during alkali-treatment of proteins. 

Mechanistic considerations (Asquith and Otterburn, 1977 Whitaker and Feeney, 1977 Friedman, 1977) suggest that added thiol or sulfite ions can inhibit lysinoalanine formation by at least three distinct mechanisms. The first is by direct competition. The added nucleophile (mercaptide, sulfite, bisulfite, thiocyanate, thiourea, etc.) can trap dehydroalanine residues derived from protein amino acid side chains, forming their respective adducts. In particular, lanthionine side chains (Figure 8)are formed from added cysteine and N-acetyl-cysteine. The second possible mechanism is... 

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