Nonenzymatic reactions, modification

Another form of post-transitional modification that may add carbohydrate to a polypeptide is nonenzymatic glycation. This reaction occurs between the reducing ends of sugar molecules and the amino groups of proteins and peptides. See Section 2.1 for further details and the reaction sequence behind this modification. 

In the analysis of proteins in forensic applications, the chemical modifications that occur to proteins, posttranslationally and nonenzymatically, are of primary importance. These chemical changes are a result of chemical reactions between side chains of the protein and reactive groups of metabolites and/or exogenous toxicants, including drugs present in extracellular fluid such as serum. The analytical accessibility of these modified proteins depends on their rate of turnover. For example, those with a slow turnover rate will be long-lived, and such problems will be much more easy to identify than those with faster turnover rates. 

This section discusses two families of structural proteins, namely, extracellular keratins and collagens. Each example represents a family of closely related chemical entities, which means dealing with complex protein mixtures. We consider two types of chemical modifications modifications arising from increased levels of glucose in body fluids (obviously related to diabetes) and those derived from acetaldehyde, the first metabolic product of ethanol ingestion. As noted, a common feature of the latter nonenzymatic modification reactions is the presence of an aldehydic functionality in the nonprotein reactant (Jelfnkovd et al 1995 Deyl and Mikgik, 1995). 

The first two steps in the synthesis of melanin are catalyzed by tyrosinase, a copper-containing oxidase, which converts tyrosine to dopaquinone. All subsequent reactions presumably occur through nonenzymatic auto-oxidation, in the presence of zinc, with formation of the black to brown pigment eumelanin. The yellow to reddish brown, high-molecular-weight polymer known as pheomelanin and the low-molecular-weight trichromes result from addition of cysteine to dopaquinone and further modification of the products. Pheome-lanins and trichromes are primarily present in hair and feathers. 

Functional properties of food protein are sensitive to changes in the size of the protein molecule, structural conformation, and the level and distribution of ionic charges. They can be modified, both enzymatically and nonenzymatically, by reactions such as protein hydrolysis, denaturation, ionization, and cross-linking. Enzymatic modifications are considered safer for food uses and therefore more desirable. However, many nonenzymatic methods have been proven safe and, because of their simplicity and great effectiveness, they are often the method of choice. Specifically, food proteins have been modi-... 

In this chapter, a review of protein deamidation and phosphorylation will be given, with emphasis on recent developments relating these reactions to use in foods. The literature on both enzymatic and nonenzymatic modifications will be included. While traditional and popular methods are summarized and remain the focus of this review, new and innovative approaches will be introduced and given special attention. 

Nonenzymatic phosphorylation with STMP appears to be able to solve many of the problems associated with POCl3. For example, STMP is an FDA-approved food additive [71], does not cause protein cross-linking, and its hydrolysis in water produces only harmless Pi. However, phosphorylation with STMP occurs at alkaline pH, which could lead to undesirable reactions and products as described earlier. STMP shows potential for food protein modification, but further research is needed to determine the exact incorporation of covalently bound Pi, to avoid alkaline pH conditions, and to move its specificity toward O-esterification rather than a mixture of O- and N-esterifica-tions. 

Thiol-containing enzymes are also critical targets for NO. The active-site thiol of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is subject to modification via NO-dependent reactions. This, in turn, leads to reaction with NAD, thus initiating nonenzymatic ADP-ribosylation reactions (Mohr... 

Several lines of evidence suggest that ADP-ribosylation in rat liver SMP can be due to nonenzymatic covalent protein modification with free ADP-ribose previously formed by enzymatic hydrolysis of NAD+. A similar reaction sequence has been proposed (3) for the mono(ADP-ribosylation) of a 30 kDa acceptor protein in beef heart SMP. The covalent modification with free ADP-ribose has the same specificity with respect to the acceptor protein as with NAD+. It is not a Schiff base adduct. Although possibly nonenzymatic, the specific mono(ADP-ribosylation) of the 30 kDa protein may fiiUfiU a physiological function in intact mitochondria. 

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