Protein hydrolytic modifications

These nonhydrolytic, post-translational enzymatic modifications of proteins, described in two recent publications (9,10), will not be treated further in this chapter. Hydrolytic modification, in vivo and in vitro, is the single most frequently occurring enzymatic modification of proteins. 

The hydrolytic modifications of proteins catalyzed by enzymes include both generalized reactions, where a relatively large number of peptide bonds are split, and limited reactions where hydrolysis of one or only a few bonds are necessary in order to achieve the desired product (see Table III). Examples of both types of reactions will be presented below. 

Some examples of in vitro hydrolytic modifications of proteins are shown in Table IV. The preparation of cheeses, chillproofing of beer, and the production of protein hydrolysates represent major uses of proteases. With the possible exception of cheese preparation, the application involves a substantial degree of hydrolysis. Therefore rather nonspecific proteases often are used. 

Table IV. In Vitro Enzyme-Catalyzed Hydrolytic Modifications of Food Proteins...

These proteases have been studied extensively because of their importance in the continuous turnover of proteins at the cellular level. The reader is referred to several recent excellent reviews (16,17,18). In this chapter, we shall concentrate on the specificity of protein turnover as an example of the potential that exists for selective hydrolytic modifications of food proteins. 

Translocation of Proteins Across Membranes. The transfer of proteins across biological membranes generally involves a hydrolytic modification step of the precursor form of the mature protein. This processing has been shown clearly to occur during segregation of secretory proteins, transport of proteins into mitochondria, and entry of plant and microbial toxins into cells as shown in Table XII. 

In this section we describe briefly the two models for inserting integral proteins into cell membranes with special emphasis on the protease(s)-catalyzed hydrolytic modification of these proteins associated with the membrane assembly process. These two models are (1) self assembly following translation of the proteins and (2) coupling of translation with insertion of the protein into the membrane. 

In mammalian cells, the two most common forms of covalent modification are partial proteolysis and ph osphorylation. Because cells lack the ability to reunite the two portions of a protein produced by hydrolysis of a peptide bond, proteolysis constitutes an irreversible modification. By contrast, phosphorylation is a reversible modification process. The phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by protein kinases, is thermodynamically spontaneous. Equally spontaneous is the hydrolytic removal of these phosphoryl groups by enzymes called protein phosphatases. 

Urethane linkages between amino groups of a protein and PEG provide a stable attachment, more resistant to hydrolytic cleavage (13). In fact, it was demonstrated on radioactively labeled PEG-derivatives that urethane links are completely stable under a variety of physiological conditions (14). The attachment of PEG to a protein via carbamate was obtained (15,16) using carbonyldiimidazole activated PEG. However, the polymer activated in this manner is not very reactive and therefore very long reaction times (48-72 h at pH 8.5) were required to achieve sufficient modifications. 

Proteins are subject to a variety of chemical modification/degradation reactions, viz. deamidation, isomerization, hydrolysis, disulfide scrambling, beta-elimination, and oxidation. The principal hydrolytic mechanisms of degradation include peptide bond... 

A host of enzymes, which are described elsewhere in the book, act on DNA and RNA. They include hydrolytic nucleases, methyltransferases, polymerases, topoisomerases, and enzymes involved in repair of damaged DNA and in modifications of either DNA or RNA. While most of these enzymes are apparently proteins, a surprising number are ribozymes, which consist of RNA or are RNA-protein complexes in which the RNA has catalytic activity. 

In addition to structure control, metal ions can act as reactive centers of proteins or enzymes. The metals can not only bind reaction partners, their special reactivity can induce chemical reaction of the substrate. Very often different redox states of the metal ions play a crucial role in the specific chemistry of the metal. Non-redox-active enzymes, e.g. some hydrolytic enzymes, often react as a result of their Lewis-acid activity [2], Binding of substrates is, however, important not only for their chemical modification but also for their transport. Oxygen transport by hemoglobin is an important example of this [3]. 

For the purpose of this chapter, enzyme-catalyzed modifications of proteins will be divided into two groups hydrolytic and nonhydrolytic reactions. Generally speaking, post-translational reactions occurring in vivo are catalyzed by highly specific enzymes under rather restricted conditions in contrast with in vitro modifications which are carried out under less specific conditions. 

The simple coordination chemistry characteristic of the majority of protein-metal interactions is replaced in certain cases by irreversible covalent modifications of the protein mediated by the metal ion. These modifications are essential for the function and are templated by the structure of the protein, as no other proteins are required for the reaction to occur. These self-processing reactions result in the biogenesis of redox cofactors in some enzymes (amine oxidases, galactose oxidase, cytochrome c oxidase) and activation of hydrolytic sites in others (nitrile hydratase). The active sites of all of these enzymes are bifunctional, directing not only the catalytic turnover reaction of the mature enzyme but the modification steps required for maturation. 

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