Phosphoproteins enzymatic dephosphorylation

The remainder of our discussion on phosphoproteins will deal with the types of enzymes involved in phosphorylation of nonenzyme proteins, substrate specificity of those enzymes, and enzymatic dephosphorylation of phosphoproteins. 

Figure 1.50 uses P-casein as an example to show that the solubihty of a phosphoprotein in the presence of calcium ions is greatly improved by partial enzymatic dephosphorylation. 

Considerations of this kind led the author to undertake an investigation using enzymes to reveal the chemical nature of phosphorus bonds that may occur in phosphoproteins. This interest came through the accidental observation that a variety of phosphomonoesterases of mammalian origin and from plants will dephosphorylate ovalbumin, a protein with a low phosphorus content. Of course, a prerequisite in the selection of the enzymes for such work is that the dephosphorylation process should not be accompanied by any other enzymatic reactions that might result from the presence of small amounts of impurities in even highly purified phosphatase preparation. in particular, an extensive proteolysis has to be excluded. 

It is important to distinguish clearly between a covalent modification for activation and an allosteric activation. A covalent modification remains with the enzyme until the modification is enzymatically reversed. For example, phosphorylase kinase and phosphorylase will remain active after phosphorylation until the enzymes are dephosphorylated as catalyzed by phosphoprotein phosphatase. In the case of allosteric activation, the enzyme will remain active as long as there is an elevated allosteric effector such as calcium. As soon as calcium is reconcentrated in subcellular fractions or subcellular organelles and/or extruded from the cell, the enzyme becomes relatively inactive because there is no longer an allosteric activator present. The same is true for allosteric inhibitors. Thus, it is advantageous that both of these mechanisms are available to the cell. 

Figure 11.16 Regulation of glycogen phosphorylase by phosphorylation/dephosphorylation The major enzymatic system in the regulation of glycogen phosphorylase (i.e. phosphorylase) by the multicyclic phosphorylation/dephosphorylation cascade is shown. Abbreviations used are Pr, protein PrK, Protein kinase phosphorylaseK, phosphorylase kinase (C2R2 where C2 and R2 are dimeric catalytic and regulatory subunits respectively) PPrP, phosphoprotein phosphatase (G), G-subunit of phosphoprotein phosphatase and p-(G), phopsho-G-subunit.

Phosphoamino acids that are part of proteins known to bind metal ions are posttranslational modifications introduced by specific protein kinases (Meggio et al, 1981 Vogel and Biidger, 1982c). The bovine milk protein casein and the hen egg-white protein ovalbumin, as well as possibly the human saliva acidic proline-iich proteins share sequence homology of their phosphorylated sites. Dephosphorylation of such sites by enzymatic phosphatase treatment usually reduces the affinity of such proteins for metal-ion binding (Bennick et al., 1981). Hence it is likely that dianionic phosphoryl moieties are directly involved in the complexation of metal ions. This seems particularly important for the two polyelectrolyte proteins that contain large amounts of phosphoserine residues, phosvitin purified from egg yolk (Ta-borsky, 1974), and the phosphoprotein purified from dentine (Linde et al, 1980). 

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