Enzymes phosphoprotein phosphatase

Control of the activity of the pyruvate dehydrogenase complex is exerted by the phosphorylation of pyruvate decarboxylase (E[), which renders it inactive. This process is catalyzed by pyruvate dehydrogenase kinase, which is always tightly bound to E[. The kinase is activated by high-energy conditions, and it requires ATP to accomplish the phosphorylation step. Another enzyme, phosphoprotein phosphatase, is weakly bound to E, and reactivates the system by removing the inhibitory phosphate group (Fig. 12-8). 

Conversion of the D form back to the I form requires action of the enzyme phosphoprotein phosphatase I. Glycogen synthase I is called the independent form of glycogen synthase, since it does not require glucose-6-phosphate for activity. 

Conversion of the D form back to the 1 form requires action of the enzyme phosphoprotein phosphatase... 

Conversely, hormones, such as insulin, which reduce cAMP levels and counteract the effects of cAMP-dependent protein kinase activity, stimulate dephosphorylation of glycogen synthase D by the enzyme phosphoprotein phosphatase I to the independent form (I) and favor glycogen synthesis. 

A few enzymes, such as the previously mentioned CNP, are believed to be fairly specific for myelin/oligodendro-cytes. There is much more in the CNS than in peripheral nerve, suggesting some function more specialized to the CNS. In addition, a unique pH 7.2 cholesterol ester hydrolase is also enriched in myelin. On the other hand, there are many enzymes that are not myelin-specific but appear to be intrinsic to myelin and not contaminants. These include cAMP-stimulated kinase, calcium/calmodulin-dependent kinase, protein kinase C, a neutral protease activity and phosphoprotein phosphatases. The protein kinase C and phosphatase activities are presumed to be responsible for the rapid turnover of MBP phosphate groups, and the PLP acylation enzyme activity is also intrinsic to myelin. 

Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. 

To serve as an effective regulatory mechanism, phosphorylation must be reversible. In general, phos-phoryl groups are added and removed by different enzymes, and the processes can therefore be separately regulated. Cells contain a family of phosphoprotein phosphatases that hydrolyze specific -Ser, -Thr, and -Tyr esters, releasing Pj. The phosphoprotein phosphatases we know of thus far act only on a subset of phosphoproteins, but they show less substrate specificity than protein kinases. 

Glucagon or epinephrine decreases [fructose 2,6-bisphosphate]. The hormones do this by raising [cAMP] and bringing about phosphorylation of the bifunctional enzyme that makes and breaks down fructose 2,6-bisphosphate. Phosphorylation inactivates PFK-2 and activates FBPase-2, leading to breakdown of fructose 2,6-bisphosphate. Insulin increases [fructose 2,6-bisphosphate] by activating a phosphoprotein phosphatase that dephosphorylates (activates) PFK-2. 

Some bacteria, including E. coli, have the full complement of enzymes for the glyoxylate and citric acid cycles in the cytosol and can therefore grow on acetate as their sole source of carbon and energy. The phosphoprotein phosphatase that activates isocitrate dehydrogenase is stimulated by intermediates of the citric acid cycle and glycolysis and by indicators of reduced cellular energy supply (Fig. 16-23). The same metabolites inhibit the protein kinase activity of the bifunctional polypeptide. Thus, the accumulation of intermediates of... 

Phosphorylation and dephosphorylation Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use adenosine triphosphate (ATP) as a phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases (Figure 5.18). 

CoA reductase activity is controlled covalently through the actions of a protein kinase and a phosphoprotein phosphatase (see Figure 18.6). The phosphorylated form of the enzyme is inactive, whereas the dephosphorylated form is active. [Note Protein kinase is activated by AMP, so cholesterol synthesis is decreased when ATP availability is decreased.]... 

The purple acid phosphatases (PAPs) are a class of phosphoprotein phosphatases which possess a p-oxo(hydroxo)-bridged dinuclear iron centre. An enzyme has been isolated from beef spleen which is purple in colour, while a violet phosphatase has been characterised from red kidney beans (KBPase). This latter enzyme consists of two subunits with M = 58200 and contains two equivalents of Zn(II) and Fe(III) per dimer which are essential for catalytic activity. KBPase hydrolyses nucleosidetriphos-phates as well as activated phosphomonoesters such as 4-nitrophenylphosphate or a-naphthyl phosphate (Beck et al., 1986). As with the beef spleen enzyme, KBPase is inhibited by tetrahedral oxoanions such PO and AsO . 

The rapid (i.e. less than 4 h) activation of TH in the median eminence by prolactin that constitutes the tonic component of prolactin stimulation does not require protein synthesis, but is probably associated with effects on the catalytic properties of this enzyme. Pasqualini and coworkers (1994) demonstrated in vitro that prolactin acts directly on TH in the mediobasal hypothalamus to trigger the phosphorylation of this enzyme. This effect, possibly mediated by protein kinase C, makes the enzyme less susceptible to inhibition by newly synthesized DA. That is, prolactin-induced short-term activation of TH results from the removal of end-product inhibition of the enzyme. Conversely, the acute reduction in TH activity measured in vitro in median eminence removed from rats 4 h after administration of bromocriptine is prevented by the coadministration of prolactin (Arbogast and Voogt, 1995). This can also be prevented by an inhibitor of phosphoprotein phosphatases, suggesting that rapid suppression of TH activity secondary to the bromocriptine-induced hypoprolactinemia may also result from dephosphorylation of the enzyme. 

Figure 6.5 Regulation of HMG-CoA reductase. HMG-CoA reductase is active in the dephospho-rylated state phosphorylation (inhibition) is catalysed by reductase kinase, an enzyme whose activity is also regulated by phosphorylation by reductase kinase kinase. Hormones such as glucagon and adrenalin (epinephrine) negatively affect cholesterol biosynthesis by increasing the activity of the inhibitor of phosphoprotein phosphatase-1, PPI-1, (by raising cAMP levels) and so reducing the activation of HMG-CoA reductase. Conversely, insulin stimulates the removal of phosphates (and lowers cAMP levels), and thereby activates HMG-CoA reductase activity. Additional regulation of HMG-CoA reductase occurs through an inhibition of synthesis of the enzyme by elevation in intracellular cholesterol levels.

In a recent communication Sundararajan and Sarma report that a phosphoprotein phosphatase from rat spleen dephosphorylates a-, /3-, and unfractionated casein (90). Since these authors state that their enzyme differs in its action from that of a phosphomonoesterase, their results are in accord with the occurrence of a variety of phosphorus bonds in proteins. In this connection it should be noted that intestinal phosphatase used in our work at pH 9.0 also liberates all of the a-casein phosphorus (72). As discussed earlier, although this enzyme at pH 6.0 hydrolyzes —N—P—... 

In this connection it is of interest to consider recent reports on enzymes specific for the hydrolysis of the phosphoprotein-phosphorus (20, 24, 27, 59, 88, 90). Although the evidence of the occurence of phosphoprotein-phosphatases is not quite unequivocal, it is striking that such enzymes are found in tissues which are abundant in phosphoproteins. Thus it is not unlikely that these phosphatases also may act as transferases. 

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 ... 

Inactivation of the enzyme phosphorylase kinase a is accomplished by dephosphorylation by phosphoprotein phosphatase. A similar phosphoprotein phosphatase is required to convert the active form glycogen phosphorylase a to the inactive glycogen phosphorylase b. It is obvious that a maximal effect of cyclic AMP-stimulated glycogen degradation is obtained when phosphorylation of phosphorylase kinase and glycogen phosphorylase is accompanied by a concomitant... 

The activity of pyruvate dehydrogenase is regulated by two mechanisms product inhibition and covalent modification (Section 6.5). The enzyme complex is allosterically activated by NAD+, CoASH, and AMP. It is inhibited by high concentrations of ATP and the reaction products acetyl-CoA and NADH. In vertebrates these molecules also activate a kinase, which converts the active pyruvate dehydrogenase complex to an inactive phosphorylated form. High concentrations of the substrates pyruvate, CoASH, and NAD+ inhibit the activity of the kinase. The pyruvate dehydrogenase complex is reactivated by a dephosphorylation reaction catalyzed by a phosphoprotein phosphatase. The phosphoprotein phosphatase is activated when the mitochondrial ATP concentration is low. 

Phosphoprotein phosphatases are responsible for the cleavage of phosphate groups from their protein acceptors. Some reports suggest that these enzymes can be inhibited by zinc. The actions of cAMP, then, can be seen to be terminated by two mechanisms the breakdown of cAMP by phosphodiesterase and the removal of phosphate groups from phosphoproteins by phosphatases. 

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