Protein 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. 

Fig. 2. Inhibition of enzymatic dephosphorylation of serine- and threonine-0-phosphate residues in proteins by toxins.

Enzymatic phosphorylation has been extensively investigated because protein phosphorylation-dephosphorylation is an important mechanism in the regulation of a variety of enzymes and proteins in mammalian cells [72-75], However, very few reports are available that describe enzymatic phosphorylation for food application. Enzymes that phosphorylate proteins are protein kinases (EC 2.7.1.37), which transfer the y-phosphate of ATP to the hydroxyl groups of serine, threonine, or tyrosine residues in proteins. Numerous protein kinases... 

Within many tissues the enzymatic activities of the pyruvate and branched chain oxoacid dehydrogenases complexes are controlled in part by a phosphorylation -dephosphorylation mechanism (see Eq. 17-9). Phosphorylation of the decarboxylase subunit by an ATP-dependent kinase produces an inactive phosphoenzyme. A phosphatase reactivates the dehydrogenase to complete the regulatory cycle (see Eq. 17-9 and associated discussion). The regulation is apparently accomplished, in part, by controlling the affinity of the protein for... 

Protein phosphatase inhibition is usually detected by colorimetric methods, but the development of a biosensor requires the search of other transduction techniques. Electrochemistry has been widely used in biosensors because of the simplicity, easy to use, portability, disposability and cost-effectiveness of the devices. As protein phosphatase is not an oxidoreductase enzyme, our work has been devoted to the investigation of novel enzymatic substrates, electrochemically active only after their dephosphorylation by the protein phosphatase. Nevertheless, colorimetric assays have been used for the optimisation of several experimental parameters. 

The system may be regarded as involving a Na+/Mg2+ co-catalysed phosphorylation step and a K+ catalysed dephosphorylation. Each phosphorylation/dephosphorylation step involves a pseudorotation of an Mg2+-stabilised 5-coordinate intermediate, resulting in transport of the alkali metal cations. The cation transport ability of the enzyme is a direct result of the enzymatic reactivity of the protein. There are three binding sites with high Na+ affinity and two with K+ affinity (occupied by Rb+ in the crystal structure determination). The structure (which is of the E2K state of the system) reveals that carboxy end of the a-subunit is held in a pocket in between transmembrane helices and acts as an unusual regulating element that controls sodium affinity and may be influenced by the membrane potential. 

FIGURE 7.44 MALDI mass spectra from on-CD analysis of the phosphopeptide-con-taining sample, (a) Peptide mass spectrum after concentration/desalting. A database search showed that the sample contained bovine protein disulfide isomerase. (b) Phosphopeptide enrichment by IMAC. Two phosphopeptides at m/z 964 and 2027 were recognized (c) Phosphopeptide enrichment followed by enzymatic on-column dephosphorylation using alkaline phosphatase. Two phosphopeptides at m/z 884 and 1947 were recognized from the mass shifts of 80 Da from (B), which were resulted from dephosphorylation [794]. Reprinted with permission from the American Chemical Society. 

The molecular basis for regulation of enzymatic activity through phosphorylation and dephosphorylation has been established in many enzyme systems (29). The significance of these reactions in histones, ribosomal proteins and KNA polymerase is not known. In an attempt to establish the specificity of the cyclic AMP-dependent protein kinases, the structure of several substrates have been determined (30). The data indicate that the sequence around the phosphorylated serine residue all contain two basic amino acids separated by no more than two residues from the N-terminal of the susceptible serine (e.g. -Arg-Arg-X-Y-Ser-). 

The metabolic functions of living organisms are maintained by a complex interplay of regulatory networks. Enzymatic activity and gene expression are permanently adapted for an optimum performance and may be completely switched on and off in a reversible manner. Typical mechanisms involved in biological systems include the stimulation and inhibition by control proteins or metabolite molecules, allosteric interactions, proteolytic activation, redox transformations, and reversible covalent bond modifications such as phosphorylation and dephosphorylation (5). 

Cyclosporine binds to an intracellular protein, cyclophilin. Cyclophilins and similar binding proteins are now referred to collectively as immunophilins and their enzymatic activities are relevant to the actions of immunosuppressants such as cyclosporine and tacrolimus. This complex inhibits the phosphatase activity of calcineurin, which in turn prevents dephosphorylation and translocation of NFAT. NFAT is required to induce a number of cytokine genes, including that for interleukin-2, which serves as a T-cell growth and differentiation factor (Krensky et al., 2005 Matsuda and Koyasu, 2000). Cyclosporine also increases expression of TGF-p, which is a potent inhibitor of IL-2 stimulated cell proliferation and generation of cytotoxic T lymphocytes (Khanna et al., 1994). 

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. 

In experiments in which a- and (3-casein are remixed in different proportions, it is noticed that if the relative concentration of the (3-casein in the mixture exceeds 20% the presence of this protein inhibits the enzymatic action, the degree of inhibition being proportional to the concentration of the (3-protein. These results may be taken as an explanation for the failure of previous investigators to dephosphorylate unfractionated casein without preceding transformation to phosphopeptones. 

Having demonstrated that the phosphorus of these two proteins can be removed enzymatically, the influence of the dephosphorylation on the proteolytic activity of pepsin and oti the pepsinogen —> pepsin transformation becomes of considerable interest. Since exposure of pepsin to pH values more alkaline than pH 6.0 results in spontaneous loss of the proteolytic activity of this protein, removal of the phosphorus had to be achieved at pH 5.6 with the aid of the potato enzyme. 

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