Enzyme modification of proteins

We have chosen to discuss enzyme modification of proteins in terms of changes in various functional properties. Another approach might have been to consider specific substrates for protease action such as meat and milk, legumes and cereals, and the novel sources of food protein such as leaves and microorganisms ( ). Alternatively, the proteases themselves provide categories for discussion, among which are their source (animals, plants, microorganisms), their type (serine-, sulfhydryl-, and metalloenzymes), and their specificity (endo- and exopeptidases, aromatic, aliphatic, or basic residue bond specificity). See Yamamoto (2) for a review of proteolytic enzymes important to functionality. 

Haard, N.F., Enzymic modification of proteins in food systems, in Chemical and Functional Properties of Food Proteins, Sikorski, Z.E., Ed., Technomic Publishing Co., Inc., Lancaster, PA, 2001, chap. 7. 

There have been a limited number of studies on the effects of enzymic modification of protein concentrates on functional properties other than solubility. Studies on functional properties, as modified by enzymic treatments, emphasize foam formation and emulsifying characteristics of the hydrolysates. Treatment of chicken egg albumen alters the functional properties of the egg proteins in terms of foam volume and stability and the behavior of the proteins in angel food cakes (25). Various proteolytic enzymes were used to degrade the egg albumen partially. However, proteolytic enzyme inhibitors indigenous to the egg proteins repressed hydrolysis of the egg proteins compared with casein. 

After their synthesis (translation), most proteins go through a maturation process, called post-translational modification that affects their activity. One common post-translational modification of proteins is phosphorylation. Two functional classes of enzymes mediate this reversible process protein kinases add phosphate groups to hydroxyl groups of serine, threonine and tyrosine in their substrate, while protein phosphatases remove phosphate groups. The phosphate-linking... 

Vitamin K is the cofactor for the carboxylation of glutamate residues in the post-synthetic modification of proteins to form the unusual amino acid y-carboxygluta-mate (Gla), which chelates the calcium ion. Initially, vitamin K hydroquinone is oxidized to the epoxide (Figure 45-8), which activates a glutamate residue in the protein substrate to a carbanion, that reacts non-enzymically with carbon dioxide to form y-carboxyglut-amate. Vitamin K epoxide is reduced to the quinone by a warfarin-sensitive reductase, and the quinone is reduced to the active hydroquinone by either the same warfarin-sensitive reductase or a warfarin-insensitive... 

Chemical modifications of proteins (enzymes) by reacting them with iV-acylimidazoles are a way of studying active sites. By this means the amino acid residues (e.g., tyrosine, lysine, histidine) essential for catalytic activity are established on the basis of acylation with the azolides and deacylation with other appropriate reagents (e.g., hydroxylamine). 

The following protocols make use of the compounds adipic acid dihydrazide and carbohy-drazide to derivatize molecules containing aldehydes, carboxylates, and alkylphosphates. The protocols are applicable for the modification of proteins, including enzymes, soluble polymers such as dextrans and poly-amino acids, and insoluble polymers used as micro-carriers or chromatographic supports. 

In earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

This class of farnesyltransferases catalyzes the posttrans-lational modification of proteins by the cholesterol precursor, farnesylpyrophosphate. One of the substrates of this enzyme is Ras. 

Chemical modification of proteins has been extensively studied over the years to identify which amino acids are involved in catalysis. Much less work has been carried out on its influence on enzyme stability. Chemical modification of proteins may yield stabilization, destabilization or no effect at all. Martinek and Berezin (1978) reported the dependence of the thermostability of chymotrypsin on the degree of alkylation of its amino groups up to 30% alkylation the stability rose slightly at 90% substitution stability increased markedly, with a maximum (110-fold) at 95% stability fell to nearly initial values when 100% amino groups were modified. (With these modifications, the optimum pH of the errzyme can change and one must therefore be cautious in comparing two different... 

Other post-translational modifications of proteins (e.g., phosphorylation) are extremely important mechanisms of regulation of enzyme activity. Very tittle work has been done on the effects of such modifications on enzyme stabilization. 

The use of proteases for modification of protein functionality is an ancient art. Originally, the enzymes were either endogenous to the food (e.g., aging of meat) or part of an added bio-system (e.g. microbial cultures in cheeses) (3). With increased knowledge of what enzymes are and how they work, their delibrate isolation and addition to food systems became widely practiced. 

Pallavicini et al. (16) utilized a-chymotrypsin immobilized on chitin to catalyze plastein formation from leaf protein hydrolyzates. When analyzed by gel exclusion chromatography, the products were comparable to those produced by soluble enzymes. Modification of Specific Functional Properties... 

---