Isopeptide formation

It may be concluded that cross-linking due to isopeptide formation is probably responsible for decreased overall digestibility of the protein (, 22) thus, if the digestive enzymes are not able to release the smaller peptides, then the question of availability of the isopeptides per se is beside the point. Ford (13) points out that the rate of digestibility of isopeptide links may preclude the maximum availability of the lysine as a result of the lysine entering the system too late to be effectively utilized by the tissue. Thus, the lysine in these linkages would be at least partly unavailable. Other workers have shown that small quantities of isopeptides are found in the urine (14). 

Recently, Jeunink and Cheftel (47) have attempted to illuminate the mechanism of extrusion texturization. They attributed the low product solubility to new disulfide bonds and non-covalent interactions, but they could not rule out a contribution due to formation of isopeptide links. The small observed increase in unavailable lysine would be consistent with isopeptide formation. 

An undesirable aspect of heat and alkaline treatment is that crosslinking compounds such as lysinoalanine are formed and can lead to severely impaired digestibility and reduced bioavailability of the protein. Such changes cannot be completely separated from the isopeptide formations (6), the Maillard reactions (2) and the racemization reactions (8) discussed elsewhere in this volume. In this chapter the chemistry, the analysis, the toxicology and the occurrence of lysinoalanine will be reviewed. 

The transglutaminases are calcium-dependent enzymes that catalyse the cross-linking of proteins by promoting the formation of isopeptide bonds between the /-carboxyl group of a glutamine in one polypeptide chain and the e-amino group of a lysine in the second (Greenberg et al., 1991). These... 

Another degradation reaction observed in suspension was the formation of covalent insulin dimers [134][136], These involve isopeptide links between two insulin molecules, that result from a transamidation reaction mainly between the B-chain N-terminus of one insulin molecule, and one of the four amide side chains in the A-chain (principally AsnA21) of the second insulin molecule. 

Heating of foods rich in proteins may lead to formation of crosslinking isopeptide bonds between the S-NH2 group of lysine and the p- and y-carboxyl groups of aspartic and glutamic acid residues or their amides. 

Kuroda, M., Ohtake, R., Suzuki, E., and Harada, T. (2000). Investigation on the formation and the determination of gamma-glutamyl-beta-alanylhistidine and related isopeptide in the macromolecula fraction of beef soup. /. Agric. Food Chem. 48, 6317-6324. 

A peptide is any compound produced by amide formation between a carboxyl group of one amino acid and an amino group of another. The amide bonds in peptides are called peptide bonds. The word peptide is usually applied to compounds whose amide bonds (sometimes called eupeptide bonds) are formed between C-1 of one amino acid and N-2 of another, but it includes compounds with residues linked by other amide bonds (sometimes called isopeptide bonds). Peptides with fewer than about 10-20 residues may also be called oligopeptides those with more residues are called polypeptides. Polypeptides of specific sequence of more than about 50 residues are usually known as proteins, but authors differ greatly on where they start to apply this term. 

Many short-lived proteins are degraded within the cytosol in ATP-dependent processes. A major process involves the small protein ubiquitin (Box 10-C).134 Once "labeled" by formation of an isopeptide linkage to ubiquitin, a peptide is attacked by proteases in the proteasome complexes (Box 7-A, Chapter 12). There it is quickly degraded. Other proteases, most of which do not require ATP, are also present in the cytoplasm (Chapter 12). How do these enzymes as well as those within the lysosomes work together to produce a harmonious turnover of the very substance of our tissues How is it possible that one protein has a long half life of many days while another lasts only an hour or two in the same cell The answer seems to be that... 

Fig. 8. Formation of isopeptide bond catalyzed by Factor XHIa. The chemical reaction was catalyzed by Factor XHIa, yielding insoluble fibrin crosslinked by Ne-(7 glutamyl) lysine bonds. Factor XIII is activated to Factor XHIa by thrombin in the presence of calcium ions and fibrin.

Each a chain contains potential glutamine acceptor sites at 221, 237, 328, and 366, and donor sites at lysine 508, 539, 556, 580, and 601 (Greenberg et al., 2003 Matsuka et al., 1996). Since the aC domains associate even in the absence of crosslinking, these interactions probably bring acceptor and donor sites in proximity, facilitating the formation of the isopeptide bonds. These bonds create a covalendy connected network of aC domains, although litde is known of its structure. In addition, there are lesser amounts of 7 trimers, tetramers, and 07 complexes. Factor XIII polymorphisms can have effects on the structure and properties of the fibrin clot (Ariens et al., 2002). 

Figure 21. General scheme for isopeptide bond formation between amino and carboxyl side groups of proteins and additional amino acids. X and Z are amino and carboxyl protecting groups, respectively Y is a carboxyl-activating group (114).

Figure 3. Structure of peptide and isopeptide bonds resulting from covalent attachment of amino acids to proteins by chemical methods. In isopeptide bond formation Rt = -CH2- or -CH2CH2- of aspartic or glutamic acid and R2 = -(CH2)n- of lysine.

Formation of internal isopeptides Titrations and peptide mapping... 

Ubiquitin is highly conserved in eukaryotes yeast and human ubiquitin differ at only 3 of 76 residues. The carboxyl-terminal glycine residue of ubiquitin (Ub) becomes covalently attached to the e-amino groups of several lysine residues on a protein destined to be degraded. The energy for the formation of these isopeptide bonds (iso because e- rather than a-amino groups are targeted) comes from ATP hydrolysis. 

Very severe heating of foods, at much higher temperatures and times than those required for sterilization, may lead to formation of isopeptide cross-links between the free NH2 group of Lys and the carboxylic group of Asp or Glu ... 

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