Enzymes by phosphorylation

Figure 9-7. Covalent modification of a regulated enzyme by phosphorylation-dephosphorylation of a seryl residue.

General Principles of Regulation of Enzymes by Phosphorylation and Dephosphorylation... 

Having shown that dibutyryl PC is monomeric under the enzyme assay conditions, we found that the phospholipase A2, which acts poorly on PE in mixed micelles, is activated by dibutyryl PC which is itself an even poorer substrate. 31p-NMR spectroscopy was employed to show that only PE is hydrolyzed in mixtures of various compositions of these two phospholipids. The fully activated enzyme hydrolyzes PE at a similar rate to its optimal substrate, PC containing long-chain fatty acid groups. Because dibutyryl PC is not incorporated into the micelles, these results are consistent with a mechanism of direct activation of the enzyme by phosphoryl-choline-containing lipids (either monomeric or micellar) rather than a change in the properties of the interface being responsible for the activation of phospholipase A2. Therefore, two functional sites on the enzyme have to be assumed an activator site and a catalytic site (6). 

Short-term hormonal regulation of ACC is achieved by covalent modifications of the enzyme by phosphorylation or dephosphorylation, which either increase or decrease its activity. These changes in enzyme activity are observed within minutes of exposure to hormones and thus are not likely due to changes in the amount of enzyme (Kim, 1983). Lee and Kim (1979) reported that incubation of rat adipocytes with epinephrine doubled the incorporation of 32P into ACC within 30 minutes and reduced enzyme activity by 61%. Witters et al. (1979) established a similar relationship between phosphorylation and inactivation of rat hepatocyte ACC following glucagon treament. 

Kinases activate a large group of lytic enzymes by phosphorylation. Lytic enzymes are localized mainly in the cytosol [133]. Their activation requires an acidic pH [134-136]. Thus proteases [137], and nucleases [138,139] are activated which start cell degradation of proteins and nucleic acids. 

Fig. 4.67. Covalent modification of an enzyme by phosphorylation, which often results in altered...

Protein kinases are a class of enzymes that change the activity of other enzymes by phosphorylating them at serine or tyrosine residues and are referred to as serine or tyrosine kinases, respectively. Serine kinases also phosphory-late some enzymes at threonine residues. Protein kinase A (a serine kinase) phosphorylates many of the enzymes in the pathways of fuel metabolism. 

The organophosphorus inhibitors have very high affinity for the active site of acetylcholinesterase. Once bound, these agents inactivate the enzyme by phosphorylation. Regeneration of the enzyme is so slow that a single dose of DFP inhibits acetylcholinesterase for over a week. 

Phosphorylation of serine residue(s) of the j8-subunit of the carboxyltransferase unit occurs in pea chloroplasts incubated in the light [20]. Alkaline phosphatase treatment reduces ACC activity in parallel to removal of phosphate groups from ACC. This activation by phosphorylation is opposite to the inhibition of animal ACC by phosphorylation but is consistent with the increase in ATP concentration and rates of fatty acid synthesis in chloroplasts in the light and the activation of other plastid enzymes by phosphorylation. These results suggest that the CT subunit reaction is rate determining for overall ACC activity, at least for the multisubunits enzyme of dicots. 

Scheme 13.1 Inactivation of an enzyme by phosphorylation, and its reactivation if not aged .

Several examples of hormonal control of enzymes by phosphorylation are considered in Chapter 4 under covalent modification of enzymes. Although protein phosphorylation-dephosphorylation may be a general regulatory mechanism for physiological processes, not all protein phosphorylation is cAMP mediated, or even hormonally mediated. 

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