Enzyme regulation reversible covalent modification

REVERSIBLE COVALENT MODIFICATION REGULATES KEY MAMMALIAN ENZYMES... 

Ghanges in the availability of substrates are responsible for most changes in metabolism either directly or indirectly acting via changes in hormone secretion. Three mechanisms are responsible for regulating the activity of enzymes in carbohydrate metabolism (1) changes in the rate of enzyme synthesis, (2) covalent modification by reversible phosphorylation, and (3) allosteric effects. 

Phosphorylation, Adenylylation, and Disulfide Reduction Lead to Reversible Covalent Modifications Allosteric Regulation Allows an Enzyme to Be... 

A reversible covalent modification that plants use extensively is the reduction of cystine disulfide bridges to sulf-hydryls. Many of the enzymes of photosynthetic carbohydrate synthesis are activated in this way (table 9.3). Some of the enzymes of carbohydrate breakdown are inactivated by the same mechanism. The reductant is a small protein called thioredoxin, which undergoes a complementary oxidation of cysteine residues to cystine (fig. 9.5). Thioredoxin itself is reduced by electron-transfer reactions driven by sunlight, which serves as a signal to switch carbohydrate metabolism from carbohydrate breakdown to synthesis. In one of the regulated enzymes, phosphoribulokinase, one of the freed cysteines probably forms part of the catalytic active site. In nicotinamide-adenine dinucleotide phosphate (NADP)-malate dehydrogenase and fructose-1,6-bis-... 

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... 

Other types of reversible covalent modification that are used to regulate the activity of certain enzymes include adenylylation (the transfer of adenylate from ATP) and ADP-ribosylation [the transfer of an adenosine diphosphate (ADP)-ribosyl moiety from NAD ]. 

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. 

There are two major ways of control. One mechanism involves reversible covalent modifications, such as phosphorylation dephosphorylation, the other requires conformational transitions by binding an allosteric ligand or regulator protein. It follows an example of regulation of an enzyme, of which the activity is subject to control by both mechanisms, then we compare the regulation of an enzyme with regulation of components of cellular signalling pathways, of which many have no enzymic activity. 

Reversible covalent modification. The catalytic properties of many enzymes are markedly altered by the covalent attachment of a modifying group, most commonly a phosphoryl group. ATP serves as the phosphoryl donor in these reactions, which are catalyzed by protein kinases. The removal of phosphoryl groups by hydrolysis is catalyzed by protein phosphatases. This chapter considers the structure, specificity, and control of protein kinase A (PKA), a ubiquitous eukaryotic enzyme that regulates diverse target proteins. 

We have seen how allosteric control can provide feedback regulation to maintain order in the body and respond to its needs. Allosteric control is reversible and the analogous type of regulation by covalent modification is the reversible modification of amino-acid side chains. Some enzymes are affected directly by both types of control. By far the most common modification of proteins is the phosphorylation of serine and threonine residues, and tyrosine phosphorylation is a key part of many control mechanisms. Protein phosphorylation is discussed below. Many other types of protein modification occursome reversible and some irreversible. 

We turn now to a different mechanism of enzyme regulation. Many enzymes acquire full enzymatic activity as they spontaneously fold into their characteristic three-dimensional forms. In contrast, other enzymes are synthesized as inactive precursors that are subsequently activated by cleavage of one or a few specific peptide bonds. The inactive precursor is called a zymogen (or a proenzyme). A energy source (ATP) is not needed for cleavage. Therefore, in contrast with reversible regulation by phosphorylation, even proteins located outside cells can be activated by this means. Another noteworthy difference is that proteolytic activation, in contrast with allosteric control and reversible covalent modification, occurs just once in the life of an enzyme molecule. 

The principal enzymes controlling glycogen metabolism—glycogen phosphorylase and glycogen synthase— are regulated by allosteric mechanisms and covalent modifications due to reversible phosphorylation and... 

In the recent literature, many examples of A/BPs containing benzophenones can be found. A first example concerns the study of HDACs. These enzymes catalyze the hydrolysis of acetylated lysine amine side chains in histones and are thus involved in the regulation of gene expression. There are approximately 20 human HDACs, which are divided into three classes (I, II, and III). Class I and II HDACs are zinc-dependent metallohydrolases that do not form a covalent bond with their substrates during their catalytic process, which is similar to MMPs. It has been found that hydroxamate 65 (SAHA, see Fig. 5) is a potent reversible inhibitor of class I and II HDACs. In 2007, Cravatt and coworkers reported the transformation of SAHA into an A/BP by installment of a benzophenone and an alkyne moiety, which resulted in SAHA-BPyne (66) [73]. They showed that the probe can be used for the covalent modification and enrichment of several class I and class II HDACs from complex proteomes in an activity-dependent manner. In addition, they identified several HDAC-associated proteins, possibly arising from the tight interaction with HDACs. Also, the probe was used to measure differences in HDAC content in human disease models. Later they reported the construction of a library of related probes and studied the differences in HDAC labeling [74], Their most... 

There are two general types of covalent modification of enzymes that regulate their activity. These are the irreversible activation of inactive enzyme precursors, the zymogens, and the reversible interconversion of active and inactive forms of an enzyme. 

The activity of enzymes (and indeed of the functionality of proteins in general) can be regulated by reversibly binding ligands (allosteric effectors) and by covalent modification (that can be either reversible or irreversible). 

Covalent modification is a major mechanism for the rapid and transient regulation of enzyme activity. Numerous enzymes of intermediary metabolism are affected by phosphorylation, either positively or negatively. Covalent phosphorylations can be reversed by a separate subclass of enzymes known as phosphatases. The aberrant phosphorylation of growth factor and hormone receptors, as well as of proteins that regulate cell division, often leads to unregulated cell growth or cancer. The usual sites for phosphate addition to proteins are the serine, threonine and tyrosine R-group hydroxyl residues. 

Posttranslational modifications are enzyme-catalyzed covalent modifications of a mature protein after it has been synthesized. Examples of posttranslational modifications are phosphorylation, glycosylation, sulfation, methylation and prenylation. Espedally those modifications that are reversible, such as phosphorylation by de-phosphorylation through the action of phosphatases are important in regulation. 

---