Bonds lysinoalanine

Figure 13.5 Formation of lysinoalanine nucleophilic additions of the e-amino group of the protein-bound lysine to the double bond of DHA residue (a) causes crosslinking of the polypeptide chain (b) lysinoalanine (c) is formed after hydrolysis.

Alkali has long been used on proteins for such processes as the retting of wool and curing of collagen, but more recently it has received interest from the food industry. Alkali can cause many changes such as the hydrolysis of susceptible amide and peptide bonds, racemization of amino acids, splitting of disulfide bonds, beta elimination, and formation of cross-linked products such as lysinoalanine and lanthionine. 

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. 

In previous papers, we have (a) reviewed elimination reactions of disulfide bonds in amino acids, peptides, and proteins under the influence of alkali (5) (b) analyzed factors that may operate during alkali-induced amino acid crosslinking and its prevention (6) (c) demonstrated inhibitory effects of certain amino acids and inorganic anions on lysinoalanine formation during alkali treatment of casein, soy protein, wheat gluten, and wool and on lanthionine formation in wool ( 7, 9) (d) demonstrated that... 

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 ... 

Figure 13. Formation of lysinoalanine in cmnamycin and duramycin. Question mark indicates the uncertainty presently existing about the number of peptide bonds between the alanine and lysine moiety in lysinoalanine.

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... 

As mentioned before, hydrolytic cleavage of peptide bonds in keratins, as well as formation of lanthionyl residues, can also occur in alkali. In addition to lanthionine, lysinoalanine [59] and beta-aminoalanine [60] residues can be formed in some keratins under alkaline conditions. 

A test to discover whether wool has been damaged in processing is to determine the fraction of the wool that dissolves under standard conditions in a solution containing urea, as a hydrogen bond breaker, and a reducing agent, usually bisulfite. Add damage increases the solubility of treated, compared with untreated wool, because add hydrolyzes peptide bonds in the protein chains. Alkali treatment decreases the solubility, because redudble cystine residue disulfide cross-links are slowly replaced by nonreducible lanthionine and lysinoalanine crosslinks in alkali (see Section 5.3.4). 

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. 

Since SH groups react more readily with double bonds than do NH2 groups (43,44,49,54), addition of thiols should trap the residue of dehydroalanine as described below. These competitive reactions should minimize lysinoalanine formation. These expectations were realized. 

Figure 12. Transformation of dehydroalanyl to lysinoalanine (LAL), S-B-(2-pyridylethyl)-L-cysteine (2-PEC), and lanthionine (LAN) residues in a protein. Hydroxide ions induce elimination reactions in cysteine and serine to form dehydroalanine. The double bond of dehydroalanine then interacts with the e-NH2 group of lysine to form LAL, with the SH group of cysteine to form LAN, and with the SH group of added 2-mercaptoethylpyridine to form 2-PEC. The latter is identical to the compound obtained from cysteine and 2-vinylpyridine.

Although proteolytic deamidation at alkaline pH was shown to be useful for increasing the surface properties of some proteins, as described earlier, proteolysis is not inevitable for other types of proteins. Furthermore, the alkaline pH condition damaged amino acid residues of protein, for instance, race-mization or the formation of lysinoalanine. Therefore, it is more desirable to use enzymes that catalyze only the deamidation without touching peptide bonds at neutral pH regions. Bacillus circulans peptidoglutaminase was used for the deamidation of proteins. The enzyme was shown to have a very limited... 

Interaction between the double bond of threonine-derived methyl dehydroalanine with the e-NH2 group of L-lysine can, in principle, produce the following methyl-lysinoalanine isomers ... 

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... 

Tables 6 and 1 compare the effects of several organic and inorganic compounds on the lysinoalanine and lysine contents of alkali-treated wheat gluten and soybean protein. These results show that all these compounds partly inhibit lysinoalanine formation. The extent of inhibition may vary from protein to protein and should be related to both the content and reducibility of the disulfide bonds (Friedman, 1978a Finley et al., 1978 a,b Masri and Friedman, 1982).

Figure 8. Inhibition of lysinoalanine (crosslink) formation. Added cysteine combines at a faster rate with the double bond of a dehydro-protein (to form a lanthionine crosslink) than does the amino group of a lysine residue (to form a lysinoalanine crosslink).

Figure 9. Inhibition of lysinoalanine formation by indirect competition. The SH group of a reduced protein combines with the double bond of a dehydro-protein, thus preventing it from reacting with the amino group of lysine to form lysinoalanine.

As already mentioned, dehydroalanine is the postulated reactive precursor for lysinoalanine. Direct evidence for dehydroalanine reactivity was obtained by Friedman et al. (1977). They showed that dehydroalanine derivatives convert lysine side chains in casein, bovine serum albumin, lysozyme, wool, or polylysine to lysinoalanine residues at pH 9 to 10. Related studies showed that protein SH groups generated by reduction of disulfide bonds are completely alkylated at pH 7.6 to lanthionine side chains. These studies demonstrate that lysinoalanine and lanthionine residues can be introduced into a protein under relatively mild conditions, without strong alkaline treatment. They also imply that it should be possible to explore nutritional and toxicological consequences of lysinoalanine and lanthionine consumption in the absence of racemiza-tion (see below). 

Additionally to the above mentioned and widely used markers, lysinoalanine (LAL) was determined in model experiments and in several commercial products. LAL is formed throughout heat and/or alkali treatment of proteins by nucleophilic reaction of the lysyl-e-amino-group with the activated double bond of dehydroalanine, which is formed by B-elimination of cystine and phosphoserine in the peptide chain. Unlike furosine, LAL crosslinking creates not only a decrease in lysine but mainly of cyst(e)ine availability in the case of alkaline treatment. 

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