Enzyme hydrolytic modifications

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 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. 

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

Phosphonate analogs to phosphate esters, in which the P—0 bond is formally replaced by a P—C bond, have attracted attention due to their stability toward the hydrolytic action of phosphatases, which renders them potential inhibitors or regulators of metabolic processes. Two alternative pathways, in fact, may achieve introduction of the phosphonate moiety by enzyme catalysis. The first employs the bioisosteric methylene phosphonate analog (39), which yields products related to sugar 1-phosphates such as (71)/(72) (Figure 10.28) [102,107]. This strategy is rather effective because of the inherent stability of (39) as a replacement for (25), but depends on the individual tolerance of the aldolase for structural modification close... 

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. 

As secretory vesicles mature, many secretory polypeptides undergo post-translational modifications. Many hormones and neuropeptides as well as hydrolytic enzymes are synthesized as inactive polypeptide precursors that need to undergo proteolysis to become active. This maturation process usually starts in the TGN and continues in the secretory vesicles, but may be completed in the extracellular space soon after exocytosis takes place in some cases. The maturation process for neuropeptides is described in Chapter 18. 

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. 

Hydrolytic activities of free and immobilized lipase were assayed by the olive oil emulsion method according to the modification proposed by Soares et al. (11). One unit of enzyme activity was defined as the amount of enzyme that liberated 1 imol of free fatty acid/min under the assay conditions (37°C, pH 7.0,150 rpm). Analyses of hydrolytic activities carried out on the lipase loading solution and immobilized preparations were used to determine the activity-coupling yield (r %), which measures the recovered enzymatic activity according to Eq. 1 ... 

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]. 

Proteases are hydrolytic enzymes that catalyze the hydrolysis of peptide bond. Alkaline proteases are most commonly used in the textile industry as additives of detergents (Gupta et al., 2002) and can be used as for the surface modification of Nylon 6,6 fibers. Nylon 6,6 is a copolymer of hexamethylene diamine and adipic acid. Many proteases such as protex Gentle L, protex 40L, protex multiplus L, and protex 50FP were used to investigate changes in the nylon 6,6 polymer. Protease... 

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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