Lysine dehydroalanine

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,... 

Beta-elimination reactions have been observed in a number of proteins. This reaction occurs primarily at alkaline pH conditions. Abstraction of the hydrogen atom from the alpha-carbon of a cysteine, serine, threonine, phenylalanine, or lysine residue leads to racemization or loss of part of the side chain and the formation of dehydroalanine (26). 

Friedman (21) studied the effect of pH on the amino acid composition of wheat gluten. At pH 10.6 and above (65 C, 3 hours) no cystine was present. LAL increased with pH above 10.6. Lysine decreased over the same range of pH s, while serine and threonine contents dropped sharply at pH 13.9. Friedman concluded that cystine is most sensitive to alkali and that LAL will form most readily if lysine residues are in proximity to the dehydroalanine formed from cystine. Thus, he explained that different steric considerations may explain the different susceptibilities of wheat gluten, casein, and lactalbumin to LAL formation. 

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). 

Table IV. Effect of Calcium Chloride Concentration on Initial Rates of p Elimination of Phosvitin and Addition of Lysine to the Dehydroalanine Formed ...

Addition Reaction. The double bond of dehydroalanine and e-methyl dehydroalanine formed by the e-elimination reaction (Equation 6) is very reactive with nucleophiles in the solution. These may be added nucleophiles such as sulfite (44). sulfide (42), cysteine and other sulfhydryl compounds (20,47), amines such as a-N-acetyl lysine (47 ) or ammonia (48). Or the nucleophiles may be contributed by the side chains of amino acid residues, such as lysine, cysteine, histidine or tryptophan, in the protein undergoing reaction in alkaline solution. Some of these reactions are shown in Figure 1. Friedman (38) has postulated a number of additional compounds, including stereo-isomers for those shown in Figure 1, as well as those compounds formed from the reaction of B-methyldehydroalanine (from 6 elimination of threonine). He has also suggested a systematic nomenclature for these new amino acid derivatives (38). As pointed out by Friedman the stereochemistry can be complicated because of the number of asymmetric carbon atoms (two to three depending on derivative) possible. 

The rate of nucleophile addition to the double bond is dependent on the nature of the nucleophile as would be expected. Finley et al. (47) have measured the relative effectiveness of the sulfhydryl group of L-cysteine and the e-amino group of a-N-acetyl-L-lysine in adding to the double bond of N-acetyl dehydroalanine. At equal concentrations of the reactive species, cysteine adds some 31 times more rapidly to the double bond than does a-N-acetyl-L-lysine (47). However, when one compares these two compounds at the same pH the relative rates in favor of cysteine (pK of sulfhydryl group = 8.15) versus a-N-acetyl-L-lysine (pK of e-amino group = 10.53) are most impressive at lower pH s (Table VI). Therefore, it has been recommended that cys-... 

The reactions of proteins in alkaline solution are very important from a number of standpoints. We have already discussed several uses of alkali treatment in food processing in the introduction. When contact between the food and alkali is kept to a minimum at the lowest temperature possible with adequate control of mixing, etc. there is presently no apparent reason to discontinue its use. Low levels of lysinoalanine occur in food which has been processed in the absence of added alkali, even at pH 6 and in the dry state (20). For example, the egg white of an egg boiled three minutes contained 140 ppm of lysinoalanine while dried egg white powder contained from 160 to 1820 ppm of lysinoalanine depending on the manufacturer (20). No lysinoalanine was found in fresh egg white, 3 Elimination and addition of lysine to the double bond of dehydroalanine reduce the level of the essential amino acid lysine. This can be prevented by adding other nucleophiles such as cysteine to the reaction. Whether lysinoalanine (and other compounds formed by addition reactions) is toxic at low levels in humans is not known. 

The pair of unshared electrons on the carbanion can undergo two reactions (a) it can recombine with a proton from the solvent to regenerate either the original amino acid side chain or its optical antipode, so that it is racemized (b) it can undergo the indicated 6-elimination reaction to form a dehydroalanine derivative, which can then combine with an e-amino group of a lysine side chain to form a lysinoalanine crosslink. 

Numerous undesirable reactions that result in organoleptic, nutritional and functional deterioration may occur in food proteins during processing and storage. These include the non-enzymatic or Maillard reactions, transamidation condensation reactions with dehydroalanine forming crosslinks, and carbonyl amine interactions, all of which may involve the free e-amino group of lysine (11,23). To minimize these reactions a significant volume of work has been done on the protective modification of the e-NH2 of lysine by formylation, acetylation, propionylation (26) or reductive dimethylation (10,11). 

Disulfide bonds present in the keratin of hair are potential binding sites for many nucleophilic molecules. Tolgyesi and Fang investigated the structural changes which occur in keratin as a result of alkaline treatments of hair. They reported that hydroxide ions initiated a p-ehmination reaction, resulting in cleavage of the disulfide bonds of cystine to produce dehydroalanine intermediates. Cysteine and lysine may react with dehydroalanine to form new cross links in keratin. Nucleophilic amines such as ethylamine and n-pentylamine may also react with dehydroalanine to form... 

HP he addition of the e-amino group of lysine across the double bond of dehydroalanine leads to a product known as lysinoalanine (Figure 1). The formation of this amino acid was initially observed in bovine pancreatic ribonuclease when the enzyme was subjected to alkaline conditions. More recently, lysinoalanine has been detected in food products... 

Reference has been made repeatedly to ,/3-unsaturated amino acids. In nisin (I) (Figure 2) and subtilin (10) (Figure 2), two residues each of dehydroalanine and one residue each of dehydrobutyrine are present. In these molecules three residues each of lysine are also found. With dehydroalanine and lysine present in the same molecules, will it be possible to verify also here the mechanistic concept of the addition of w-amino groups of amino acids across the double bond of ,/ -unsaturated amino acids (Figure 10) and demonstrate the formation of lysinoalanine ... 

When nisin, fragments of nisin (still containing dehydroalanine and lysine), and subtilin were treated under basic conditions, the formation of lysinoalanine was observed in each case. Treatment, for instance, of the carboxyl-terminal fragment of nisin (Figure 11) under these conditions (11) (IN N-ethylmorpholine, pH 10.65, 7 days, room temperature) followed by total hydrolysis and amino acid analysis showed the presence... 

Figure 10. Formation of lysinoalanine from lysine and dehydroalanine...

Not for one moment must we, however, overlook the prerequisite in the form of the presence of dehydroalanine for the formation of lysinoalanine. Do all residues of the ,/3-unsaturated amino acid generated react by way of /3-addition with e-amino groups of lysine (or with other nucleophiles) Which are the physiological effects of ,/3-unsatu-rated amino acids in proteins of food products of mans daily diet The answers to these questions are largely unknown. 

Lysinoalanine is formed by -elimination reaction of cystine-cysteine and serine with the formation of dehydroalanine and the subsequent addition of the e-amino group of lysine across the reactive C—C double bond (30). The formation of other amino acids such as ornithinoalanine (31), lanthionine (32), and /3-aminoalanine (33) by similar mechanism has been described. Gross et al. (34) have pointed out that lysinoalanine... 

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. 

In 1964, Patchornik and Sokolovsky 9) observed the formation of an unusual amino acid when they treated S-dinitrophenylated ribonuclease with alkali. They concluded that the new amino acid was the result of an addition reaction between the e-amino group of lysine and dehydroalanine. Later the same year, Bohak (10) reported the isolation of a new amino acid (N-e-)D,L-2-amino-2-carboxyethyl)-L-lysine, from the acid hydrolyzte of alkaline treated proteins. The trivial name lysinoalanine (LAL) was assigned to the new amino acid. Patchornik and Sokolovsky (11) reported the importance of cystine in the formation of dehydroalanine in proteins. The observation was important in that dehydroalanine is an immediate precurser of LAL. Ziegler reported that alkaline treatment of wool also resulted in the formation of LAL and later found ornithinoalanine as well as LAL in alkaline-treated silk protein (12). Bohak (10) reported that LAL was... 

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. 

The reaction of lysine with dehydroalanine was studied by Snow et al. (21) using the N-a-acetyl-dehydroalanine methyl ester as a model. The studies with the model compound are difficult to compare with reactions that occur in proteins because proteins are such complex molecules and analysis is difficult. The comparison of the variety of proteins which form LAL suggests that although dehydroalanine may be formed by a variety of pathways, the Michael addition between lysine and dehydroalanine is rapid, particularly at higher pHs. 

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. 

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