Phosphorylation enzyme activity affected

Protein phosphorylation-dephosphorylation is a highly versatile and selective process. Not all proteins are subject to phosphorylation, and of the many hydroxyl groups on a protein s surface, only one or a small subset are targeted. While the most common enzyme function affected is the protein s catalytic efficiency, phosphorylation can also alter the affinity for substrates, location within the cell, or responsiveness to regulation by allosteric ligands. Phosphorylation can increase an enzyme s catalytic efficiency, converting it to its active form in one protein, while phosphorylation of another converts it into an intrinsically inefficient, or inactive, form (Table 9—1). 

Several key questions remain with regard to the regulation of tyrosine hydroxylase by phosphorylation. What is the precise effect of the phosphorylation of each of these serine residues on the catalytic activity of the enzyme How does the phosphorylation of multiple residues affect enzyme activity Does the phosphorylation of one residue affect the ability of the others to be phosphorylated Tyrosine hydroxylase provides a striking example as to how multiple intracellular messengers and protein kinases converge functionally through the phosphorylation of a single substrate protein. Phosphorylation of tyrosine hydroxylase by cAMP-dependent and Ca2+-dependent protein kinases and by MAPK cascades... 

Phenolic compounds naturally occurring in plants have induced many physiological responses that duplicate those reported for ozone and/or peroxyacetylnitrate (PAN). Chlorogenic acid is a competitive inhibitor of lAA-oxidase (35) and plant growth is adversely affected by increased concentrations of auxins (36). Concentrations of chlorogenic acid are increased in tobacco tissue exposed to ozone ( ) Phenols inhibit ATP synthesis (37), oxidative phosphorylation ( ) and SH enzyme activity (27) they increase respiration (38), reduce CO2 fixation (22), modify both membrane permeability (40) and oxidation rate of reduced NADH... 

Phosphorylation of an enzyme can affect catalysis in another way by altering substrate-binding affinity. For example, when isocitrate dehydrogenase (an enzyme of the citric acid cycle Chapter 16) is phospho-rylated, electrostatic repulsion by the phosphoryl group inhibits the binding of citrate (a tricarboxylic acid) at the active site. 

Activity is modulated by other proteins present in the membrane. These include a glycoprotein (MW 53 000) which stimulates ATPase activity 138 a 60 000 molecular weight protein, which is phosphorylated in a calmodulin-dependent fashion, affects accumulation of calcium 139 while the activity of the enzyme is affected by an endogenous kinase and phosphatase which phosphorylates and dephosphorylates the protein.140 Phospholamban is a proteolipid (MW 22 000) in cardiac SR which undergoes both cyclic AMP-dependent and calcium-calmodulin-dependent phosphorylation,141 but at different sites. All these proteins are probably involved in regulating the activity of the calcium pump. 

Fatty add synthetase is not controlled directly by phosphorylation however, insulin, glucagon, and thyroxine have an effect on its activity by controlling its cellular concentration. Both insulin and thyroxine increase the biosynthesis of the enzyme, whereas glucagon is inhibitory. Thyroxine and glucagon appear to regulate the biosynthesis at the transcription level, whereas insulin affects the enzyme activity at the translation level. It has no effect on cellular fatty add synthetase mRNA concentration. In summary, fatty add synthetase levels are up in the fed state and down in the fasting state. 

Glucagon affects hepatic lipid metabolism. A major effect is inhibition of fatty acid synthesis, which is mainly due to the phosphorylation and inhibition of acetyl-GoA carboxylase by cAMP-dependent protein kinase. ATP-citrate lyase is also phosphorylated, but it is unclear that this is involved in the inhibition of lipogene-sis. Glucagon also inhibits cholesterol synthesis apparently due to a decrease in the activity of hydroxymethylglutaryl-CoA reductase. This is thought to result from a decrease in the activity of protein phosphatase I due to the increased phosphorylation and activation of a heat stable inhibitor by cAMP-dependent protein kinase. This mechanism could also contribute to the effects of glucagon on other hepatic enzymes. 

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. 

N is often limiting in the marine environment. Further, many enzymes are sensitive to cellular substrate concentrations rather than extracellular concentrations and it is difficult to measure the relevant intracellular metabohte pools. In vitro assays may affect the conformation of enzymes and the degree to which they are modified. For example, allosteric effects (see Section 1.3.3) may be modified under in vitro conditions. Many enzymes undergo posttranslational regulation wherein enzyme activity is affected by binding of activator/inactivator proteins and covalent modification of the enzyme (e.g., adenylylation, phosphorylation or carbamylation) (Ottaway, 1988). When there is posttranslational modification of enzymes, enzyme activity measured in assays may be unrelated to in vivo activity (see Section 2.2.1) and there are few ways to determine the extent of enzyme modification in nature. 

Anticholinesterase insecticides phosphorylate the active site of cholinesterase in all parts of the body. Inhibition of this enzyme leads to accumulation of acetylcholine at affected receptors and results in widespread toxicity. Acetylcholine is the neurohormone responsible for physiologic transmission of nerve impulses from preganglionic and postganglionic neurons of the cholinergic (parasympathetic) nervous system, preganglionic adrenergic (sympathetic) neurons, the neuromuscular junction in skeletal muscles, and multiple nerve endings in the central nervous system (Fig. 10-5). 

The protein kinase A serves to phosphorylate a set of tissue-specific substrate enzymes, thereby affecting their activity. 

Acute intoxication by anti-ChE agents causes muscarinic and nicotinic signs and symptoms, and, except for compounds of extremely low lipid solubility, affects the CNS. Systemic effects appear within minutes after inhalation of vapors or aerosols. The onset of symptoms is delayed after GI and percutaneous absorption. Duration of effects is determined largely by the properties of the compound lipid solubility, whether it must be activated to the oxon, stability of the organophos-phorus-AChE bond, and whether aging of phosphorylated enzyme has occurred. 

Am. The hormones epinephrine and glucagon cannot penetrate cell membranes. They affect metabolic processes by binding to specific receptors on the membrane, which receptors in turn activate a specific enzyme bound to the inner membrane surface, adenylate cyclase. This enzyme converts ATP to cyclic AMP (cyclic adenosine monophosphate), or c-AMP. The presence of c-AMP activates another enzyme, protein kinase, which phosphorylates and activates phosphorylase kinase. Phosphorylase kinase phosphorylates phosphorylase b (inactive) to form phosphorylase a (active) which in turn cleaves glucose from glycogen by phosphorolysis to yield glucose-I-PO4. 

A possible link between the effects of Cr(III) and Cr(VI) on carbohydrate metabolism and enzyme activation has been overlooked in the studies of Cr(III) as a nutrient (5). Treatment of rats with large doses of Cr(VI) (Na2Cr20v 20-40 mg kg subcutaneously) induced a severe, but short term, decrease in blood insulin levels (624). A significant insulin-independent stimulation of 3-0-methylglucose uptake by isolated rat adipocytes was achieved in the presence of Na2Cr207 [50-300 pM Cr(VI)] this effect was strictly ATP dependent, which implies a relationship to phosphorylation reactions (625). A detailed study of this effect by Yurkow and Kim (295, 626) showed that treatment of intact rat hepatoma cells with Cr(VI) (100 pM) induced the insulin-independent activation of certain types of protein kinases, which led to increased phosphorylation levels in various proteins these phosphorylated proteins could then participate in insulin signaling pathways. Treatment with Cr(VI) did not affect the insulin-dependent phosphorylation on p-subunits of insulin receptors. Thus, Cr(VI), similar to V(V) (618) but unlike the biologically active Cr(III) complexes (496, 497, 618), acts as an insulin mimetic rather than potentiator. Unlike V(V), however, the action of Cr(VI) resulted from kinase activation rather than... 

It should be noted that during 3 hours of incubation with Pi, about 4800 cpm were incorporated although enzyme activity was not affected at all. This observation indicates that the phosphorylation which occurred in the absence of epinephrine has no functional relationship to enzyme activity. The phosphorylation which occurs in the presence of epinephrine, however, can inactivate carboxylase. Indeed, this phosphorylation of the carboxylase which is unrelated to enzyme activity may have misled some to conclude that the covalent phosphorylation of carboxylase has no physiological significance (25,39,99). Witters al. (129) have also provided evidence for the causal relationship between the phosphorylation and inactivation of rat liver acetyl-CoA carboxylase following glucagon treatment of isolated hepatocytes which had been prelabeled with Pi. 

---