Enzyme noncovalent modification

N-Myristoylation is achieved by the covalent attachment of the 14-carbon saturated myristic acid (C14 0) to the N-terminal glycine residue of various proteins with formation of an irreversible amide bond (Table l). 10 This process is cotranslational and is catalyzed by a monomeric enzyme called jV-myri s toy 11ransferase. 24 Several proteins of diverse families, including tyrosine kinases of the Src family, the alanine-rich C kinase substrate (MARKS), the HIV Nef phosphoprotein, and the a-subunit of heterotrimeric G protein, carry a myr-istoylated N-terminal glycine residue which in some cases is in close proximity to a site that can be S-acylated with a fatty acid. Functional studies of these proteins have shown an important structural role for the myristoyl chain not only in terms of enhanced membrane affinity of the proteins, but also of stabilization of their three-dimensional structure in the cytosolic form. Once exposed, the myristoyl chain promotes membrane association of the protein. 5 The myristoyl moiety however, is not sufficiently hydrophobic to anchor the protein to the membrane permanently, 25,26 and in vivo this interaction is further modulated by a variety of switches that operate through covalent or noncovalent modifications of the protein. 4,5,27 In MARKS, for example, multiple phosphorylation of a positively charged domain moves the protein back to the cytosolic compartment due to the mutated electrostatic properties of the protein, a so-called myristoyl-electrostatic switch. 28 ... 

The most common way of regulating metabolic activity is by direct control of enzyme activity. Enzyme activities are usually regulated by noncovalent interaction with small-molecule regulatory factors (see chapter 9) or by a reversible covalent modification, such as phosphorylation or... 

High hydrostatic pressure induces changes in protein conformation, solvation and enzyme activities via reversible and non-reversible effects on intra- and inter-molecular interactions (noncovalent bonds) [1]. To have access to these structural modifications, spectroscopic investigations are required which necessitate special spectroscopic adaptations. Two improvements are presented first for enzyme reactions and second for structural determination. 

Two mechanisms that are commonly employed in altering enzyme activity are covalent modification and allosteric regulation. Covalent modification is an enzymatically catalyzed reaction that involves the reversible formation of a covalent bond between a small molecule and a specific amino acid side chain(s) on an enzyme that affects its activity. Allosteric regulation of an enzyme s activity involves noncovalent binding of a small molecule at a site other than the active site that alters the enzyme s activity. Unlike the limited examples of covalent modification that have been discovered (see Table 15-1), a wide variety of small molecules have been found to regulate the activity of particular enzymes allosterically. 

The above definition of molecular chaperone is entirely fnnctional and contains no constraints on the mechanisms by which different chaperones may act. The term noncovalent is nsed to exclude those proteins that carry out posttranslational covalent modifications. Protein disulfide isomerise may seem to be an exception, bnt it is both a covalent modification enzyme and a molecular chaperone. It is helpful to think of a molecnlar chaperone as a fnnction rather than as a molecnle. Thns, no reason exists why a chaperone function shonld not be a property of the same molecnle that has other fnnctions. Other examples include peptidyl-prolyl isomerase, which possesses both enzymatic and chaperone activities in different regions of the molecnle, and the alpha-crystallins, which combine two essential fnnctions in the same molecnle in the lens of the eye-contribnting to the transparency and the refractive index reqnired for vision as well... 

Enzyme activity can be regulated by covalent modification or by noncovalent (allosteric) modification. A few enzymes can undergo both forms of modification (e.g., glycogen phosphorylase and glutamine synthetase). Some covalent chemical modifications are phosphorylation and dephosphorylation, acetylation and deacetylation, adeny-lylation and deadenylylation, uridylylation and deuridyly-lation, and methylation and demethylation. In mammalian systems, phosphorylation and dephosphorylation are most commonly used as means of metabolic control. Phosphorylation is catalyzed by protein kinases and occurs at specific seryl (or threonyl) residues and occasionally at tyrosyl residues these amino acid residues are not usually part of the catalytic site of the enzyme. Dephosphorylation is accomplished by phosphoprotein phosphatases ... 

Noncovalent forces have also been reported to play an important role in these enzyme-catalyzed reactions [50,56]. Moreover, Hofsten and Lalasidis [50] were of the opinion that covalent forces did not play a role in these reactions. Several investigators have shown that the product produced during the plastein reaction is composed of aggregates held together by hydrophobic and ionic bonds [57,58]. Others [59] emphasized an entropy-driven aggregation process. Trans-peptidation has been considered by a number of authors [46,60,61] as the mechanism of enzymatic modification processes (resynthesis, plastein reaction, EPM). That means that a great number of peptide bonds are split and new covalent bonds formed in the course of the enzymatic process. 

Catalytic activity The regulation of enzyme activities is achieved in two modes, namely switching on-and-off and tuning up-and-down. Covalent modifications of enzymes effectively switches their activities on or off, whereas noncovalent effectors tune enzyme activities up or down by affecting their kinetic parameters. 

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