Phosphoprotein Enzymes

Enzymes which catalyse phosphate transfer may themselves be phosphoproteins. Phosphorylating enzymes have been tentatively divided into three types  

Those where the extent of phosphorylation regulates their activity 

Those which form phosphorylated intermediates during their action 

Those which contain phosphorylated residues as part of their structure 

Glycogen phosphorylase (14.48) mw = 370,000, exists in active (A) and relatively inactive (B) forms which differ in quaternary structure and phosphate content. The active phosphorylase (A) contains two phosphate groups bound to two serine units, out of a total of 30 serine units in each protein molecule. This enzyme is hydrolysed by phosphorylase phosphatase to produce the inactive (B) form which is devoid of phosphate groups. Re-phosphorylation to produce the active (A) form can be achieved with ATP and phosphorylase kinase. 

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The data discussed above provide evidence for the existence of two conformations with different capacities and orientations of cation sites. In the Ei form of Na,K-ATPase, the exposure of C3(T,i) to cleavage reflects that the cation sites of the phosphoprotein are in an inward oriented conformation with a capacity for occlusion of 3Na ions. The E2 form with exposed T1 and protected C3(T3) occludes either 2Na or 2Rb (K ) in the phosphoform or 2Rb (K ) in the unliganded enzyme. 

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. 

Ser/Thr-protein phosphatases are ubiquitous enzymes which constitute the catalytic domains of multiprotein complexes. They are responsible for the dephosphorylation of a range of phosphoproteins. Several protein phosphatases have been characterized by X-ray crystallography and display an active site structure similar to purple acid phosphatase. 

Phosphoproteins.—A chemical synthesis of partially and fully phosphorylated protamines has been described during the past year,98 and structural requirements for the enzymatic phosphorylation of phosvitin have been delineated.99 Phosphorylated forms of phosphofructokinase100 and fatty acid synthetase101 have been discovered recently both may be concerned with the regulation of their respective enzymes. 

Phosphoglucomutase was isolated by the Coris and crystallized by Najjar. Its mechanism of action was suggested from experiments by Leloir in 1951 using [32P]. The enzyme is a phosphoprotein. Leloir showed that the phosphate group was transferred from the enzyme to G-1-P in the course of the reaction to give G-1,6 diP, which then donated the phosphate from its 1-position back to the enzyme, releasing G-6-P ... 

This enzyme [EC 2.7.1.123], also referred to as calcium/ calmodulin-dependent protein kinase type II, and micro-tubule-associated protein MAP2 kinase, catalyzes the reaction of ATP with a protein to produce ADP and an 0-phosphoprotein. The enzyme requires calcium ions and calmodulin. Proteins that can serve as substrates include vimentin, synapsin, glycogen synthase, the myosin light-chains, and the microtubule-associated tau protein. This enzyme is distinct from myosin light-chain kinase [EC 2.7.1.117], caldesmon kinase [EC 2.7.1.120], and tau-protein kinase [EC 2.7.1.135]. 

This set of enzymes [EC 3.1.3.16], also known as serine/ threonine-specific protein phosphatases, catalyzes the hydrolysis of a seryl- or threonyl-bound phosphate group from a wide range of phosphoproteins, including a number of enzymes which have been phosphorylated under the action of a kinase. Thus, phosphoprotein J- water yields protein J- orthophosphate. 

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 ... 

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. 

Measurements of the steady state phosphoprotein level at different temperatures revealed that phosphoprotein formation is accompanied by a large and constant enthalpy change of 48 kJ/mol. In contrast, the likewise quite high activation energy of phosphoprotein formation exhibits a pronounced break between 20°C and 30°C. A break in the Arrhenius plot of the calcium-dependent ATPase has been observed in the same temperature range and has been interpreted as transitions between two activity states of the enzyme. Apparently, the phosphorylation of the calcium free protein by inorganic phosphate exhibits a similar kind of activity transition as observed for the calcium-dependent interaction of the transport protein with ATP131. A similar transition phenomenon complicates the time course of phosphoprotein formation... 

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.]... 

Casein is not coagulated by heat. It is precipitated by acids and by rennin. a proteolytic enzyme obtained from the stomach or calves. Casein is a conjugated protein belonging lo the group uf phosphoproteins. The enzyme trypsin can hydrolyze off a phosphorus-containing peptone. 

While it might appear that the phosphoprotein obtained in the above labeling experiments confirms the Morton hypothesis, the pH relationships require some explanation, and various other considerations such as the potent inhibitory properties of phosphate at pH 8.0, its poor inhibitory properties at pH 5.0, the extreme thermodynamic stability of the phosphoprotein and other thermodynamic considerations require further demonstration of a phosphoryl enzyme intermediate (116, 119,... 

With the establishment of the phosphoryl enzyme, the question was whether or not the phosphoryl enzyme was the same as the phospho-protein found by incubating inorganic phosphate with alkaline phosphatase at low pH (35, 114-116, 119, 120). Wilson and Dayan (105) pointed out that the phosphoprotein is thermodynamically very stable It is 105 times more stable than O-phosphorylserine (125) and 0-phosphoryl ethanolamine (105, 126). Alkaline phosphatase, as a true catalyst, must catalyze both the hydrolysis and the formation of phosphate esters. Therefore, if a serine residue existed which was capable of forming a thermodynamically stable phosphate ester, alkaline phosphatase as a nonspecific catalyst would catalyze its formation from both inorganic phosphate and phosphoester substrates. 

A plot of (E°)/(E-P) vs. l/(Pi) should yield a straight line whose slope is Kk-P and whose intercept is KE-p/Ki. These results are seen in Table VIII, where the results from the two different approaches compare quite well, indicating that the phosphoryl enzyme and the phosphoprotein are the same. These data also show that the reason that little phosphoryl enzyme is detected by phosphate labeling at pH 7.0 and above is not because the phosphoryl enzyme is unstable... 

Most mechanisms for the (Ca2+, Mg2+)-ATPase are modifications of the proposal of deMeis et a/.128 An example is shown in Figure 9. The ATPase can utilize CaATP and MgATP as substrates.147 Two moles of Ca2+ are bound per mole of the phosphorylation site with high affinity, followed by phosphorylation of the enzyme by ATP. Conformational changes in the phosphoprotein lead to the calcium sites being accessible to the intravesicular space, with decrease in their affinity for... 

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