Isopeptide hydrolysis

It now appears more likely that gut enzymes may be responsible for partial isopeptide hydrolysis (22). Waibel and Carpenter (11) suggest that hydrolysis may occur within the intestinal wall after absorption of the isopeptide. However, these studies on absorption of the free e(Y-L-glutamyl)-L-lysine may not be relevant to cross-links in an overheated protein. 

This enzyme [EC 3.4.19.5], now known as /3-aspartyl-peptidase, is a mammahan cytosolic protein that catalyzes the hydrolysis of a /3-hnked aspartyl residue from the N-terminus of a polypeptide. Other isopeptide linkages (e.g., a y-glutamyl hnkage) are not hydrolyzed by this enzyme. 

Such isopeptides also have been found naturally present in keratin (77,78,85) and in polymerized fibrin (79). Their quantitative determination requires an enzymic hydrolysis using pepsin, pronase, amino-peptidase, and prolidase as described by Cole et al. (135) followed by a chromatographic separation using an amino acid analyzer under very specific conditions (80). 

The delay is probably due to the late absorption of the isopeptide (in the distal part of the small intestine) while free lysine is absorbed in the duodenum. After its late absorption, the isopeptide is transported readily to the kidneys which hydrolyze it very rapidly (as indicated by the kinetics of the expiration of 14C02 after intravenous injection) (see Figure 4). Since no free isopeptide was found in the urines, this hydrolysis is complete. 

Hydrolysis of the Isopeptide Bond. The bio availability of methionine covalently linked to casein has been studied by the response (protein efficiency ratio) of rats that have been fed the modified protein (23,63). The covalently bound methionine appears to be as available as the free amino acid. This finding indicates that there is an efficient enzymatic... 

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. 

Furthermore, enzymatic hydrolysis of model isopeptides N -oligo(L-methionyl)-l-lysine from Bio-beads13031 by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products 3041. 

The function of the isopeptides in the various tissues is not clear, although they may contribute directly to the structure of the particular protein. For example, prior to keratinization or by simply increasing the molecular weight of a protein system confers enhanced stability, decreased solubility and increased resistance to enzymic hydrolysis. 

The major problem in all work carried out on isopeptides was related to the fact that the isopeptide bond is chemically an amide bond and as a consequence of this is susceptible to attack by acids or alkalis, thus destroying the isopeptide. The only possible methods were microbiological or enzymic, both of which obviate the problem of random hydrolysis. Methods of enzymic digestion had previously been knowi and adequately used however, such methods, although suitable for globular proteins, proved to... 

The higher esterification rate of P- and y-carboxyl groups can be used for selective reactions. On the other hand the P- and y-carboxyl groups are more rapidly hydrolyzed in acid-catalyzed hydrolysis since protonation is facilitated by having the ammonium group further away from the carboxyl group. Alkali-catalyzed hydrolysis of methyl or ethyl esters of aspartic or glutamic acids bound to peptides can result in the formation of isopeptides. 

These isopeptide bonds are cleaved during acidic hydrolysis of protein and, therefore, do not contribute to the occurrence of unusual amino acids. A more intensive heat treatment of proteins in the presence of water leads to a more extensive degradation. 

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