Enzymes covalent modulation

Other regulatory enzymes are modulated by covalent modification of a specific functional group necessary for activity. The phosphorylation of specific amino acid residues is a particularly common way to regulate enzyme activity. 

The mode of action of enzymes, be they allosteric or covalently modulated (441), is a topic of major importance and no mean complexity. It is not our intention here to cite other than a few principles and examples in relation to the way in which solid-state NMR promises to elucidate the vast, ramifying... 

In allosteric enzymes, the activity of the enzyme is modulated by a non-covalently bound metabolite at a site on a protein other than the catalytic site. Normally, this results in a conformational change, which makes the catalytic site inactive or less active. Covalent modulated enzymes are interconverted between active and inactive forms by the action of other enzymes, some of which are modulated by allosteric-type control. Both of these control mechanisms are responsive to changes in cell conditions and typically the response time in allosteric control is a matter of seconds as compared with minutes in covalent modulation. A third type of control, the control of enzyme synthesis at the transcription stage of protein synthesis (see Appendix 5.6), can take several hours to take effect. 

FIGURE 142.5 Proposed mechanism of psoralen-fatty-acid adduct action on protein kinase C (PKC). UV-A irradiation induces a covalent adduct between psoralen and phospholipids that stimulates phospholipase A2. The enzyme hydrolizes the photocompound, producing psoralen-fatty-acid adduct, which in turn, activates protein kinase C. The activation of this phosphorylating enzyme can modulate cell function and metabolism, i.e., stimulating melanogenesis in melanocytes. 

The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through non-covalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). 

One of the most-studied covalent modifications is the acetylation of the lysine residues of histone tails. The acetylation state of lysines of nucleosomal histones modulates chromatin structure and regulates gene transcriptional activity. The balance of lysine acetylation is controlled by the antagonistic action of two enzyme families histone deacetylases (HDACs) and histone acetyltransferases (HATs). In humans there are essentially three main HDAC subclasses [6]. 

As indicated in Section 6.3.3 and Table 6.2 the key control step is mediated by glycogen phosphorylase, a homodimeric enzyme which requires vitamin B6 (pyridoxal phosphate) for maximum activity, and like glycogen synthase (Section 6.2) is subject to both allosteric modulation and covalent modification. 

Once assembly of a mature peptide has been completed by the NRPS, the product remains covalently linked to the PCP domain of the last module as a thioester. Release into solution from the assembly line is accomplished by a variety of enzyme-catalyzed reactions as described below (Figure 8). 

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 ... 

In another important class of regulatory enzymes, activity is modulated by covalent modification of the enzyme molecule. Modifying groups include phosphoryl, adenylyl, uridylyl, methyl, and adenosine diphosphate ribosyl groups (Fig. 6-30). These groups are generally linked to and removed from the regulatory enzyme by separate enzymes. 

Langoth et al. [86] studied the properties of matrix-based tablets containing the novel pentapeptide leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) that has been shown to have pain-modulating properties. The matrix-based tablets were made with the thiolated polymer PCP. The covalent attachment of cysteine to the anionic polymer PCP leads to an improvement of the stability of matrix tablets, enhances the mucoadhesive properties, and increases the inhibitory potency of PCP towards buccal enzymes. All these factors lead to stability of the peptide and a controlled drug release for the peptide was obtained for more than 24 h. Also, the tablets based on thiolated PCP remained attached on freshly excised porcine mucosa 1.8 times longer than the corresponding unmodified polymer. 

Figure 10.4 Examples of covalent capture methods. (A) Covalent modification of native cysteines has been shown to modulate ion-channel activity and in the case of enzymes lead to allosteric inhibition. These findings can be important for structural-functional characterization or as starting points for drug discovery. (B) Reversible covalent capture using imine chemistry.

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