Regulation of Glycogen Phosphorylase by Phosphorylation

Glycogen phosphorylase was the first enzyme shown to be regulated via protein phosphorylation (Krebs, 1959). In recognition of their trad-blazing work, Edwin Krebs and Edmond Fisher were rewarded the Nobel prize for Chemistry in 1992. 

In the framework of the symmetry model (see 2.3), a T- and R-form can be formulated for glycogen phosphorylase. In the T-form glycogen phosphorylase binds its substrates and activating effectors with lower affinity, while in the R-form it possesses higher affinity for substrates and activating effectors. 

High resolution X-ray structures of the a- and b- form of rabbit muscle phosphorylase permit a view into some of the structural differences of the various allosteric forms of the enzyme. Furthermore, the data give an impression of the mechanism of binding of effectors and the influence that phosphorylation has on substrate binding and enzyme activity (Barford et al., 1991). The following discussion will be restricted to the observed consequences of phosphorylation. 

Phosphorylation of glycogen phosphorylase is the initiator for the coupled conformational changes, which are coimmmicated over a large distance to the active site. Similar to the allosteric mechanism of phophofructokinase, the inter-subunit contact sinfaces play a decisive role for the communication between the phosphorylation site 

Isocitrate dehydrogenase catalyzes the NAD-dependent reduction of isocitrate to a-ketoglutarate. The dimeric enzyme is regulated via phosphorylation. Phosphorylation on SerllS leads to a complete inactivation of the enzyme. 

B) Simplified schematic drawing showing changes in the N-terminus of rabitt muscle glycogen phos-phorylase.The N-terminal tail is shown as a thick black line. The catalytic site Cat is on the far side of the molecule to the viewer. 

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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.

Figure 11.15 Regulation of glycogen synthase by phosphorylation/dephosphorylation The active fonn of glycogen synthase a is the dephosphorylated form which is inactivated by the phosphorylation of two Ser. Glycogen synthase is regulated by the monocyclic phosphorylation/ dephosphorylation cascade in a manner reciprocal to that of glycogen phosphorylase.

Fig. 7.18. Regulation of glycogen metabolism in muscle. Phosphorylase kinase stands at the center of regulation of glycogen metabolism. Phosphorylase kinase may exist in an active, phosphorylated form and an inactive, unphosphorylated form. Phosphorylation of phosphorylase kinase is triggered by hormonal signals (e.g. adrenahne) and takes place via an activation of protein kinase A in the cAMP pathway. In the absence of hormonal stimulation, phosphorylase kinase can also be activated by an increase in cytosolic Ca. The active phosphorylase kinase stimulates glycogen degradation and inhibits glycogen synthesis, in that, on the one side, it activates glycogen phosphorylase by phosphorylation, and on the other side, it inactivates glycogen synthase by phosphorylation.

FIGURE 6-31 Regulation of glycogen phosphorylase activity by covalent modification. In the more active form of the enzyme, phosphorylase a, specific Ser residues, one on each subunit, are phosphorylated. Phosphorylase a is converted to the less active phosphorylase b by enzymatic loss of these phosphoryl groups, promoted by phosphorylase phosphatase. Phosphorylase b can be reconverted (reactivated) to phosphorylase a by the action of phosphorylase kinase. 

The control of glycogen phosphorylase by the phosphorylation-dephosphorylation cycle was discovered in 1955 by Edmond Fischer and Edwin Krebs50 and was at first regarded as peculiar to glycogen breakdown. However, it is now abundantly clear that similar reactions control most aspects of metabolism.51 Phosphorylation of proteins is involved in control of carbohydrate, lipid, and amino acid metabolism in control of muscular contraction, regulation of photosynthesis in plants,52 transcription of genes,51 protein syntheses,53 and cell division and in mediating most effects of hormones. 

Regulation of glycogen synthase by multisite phosphorylation. The location of phosphorylation sites ( ) and the protein kinases that phosphorylate at these sites (boxes) are shown. Phosphorylations occur only at N- and C-terminal regions of the enzymes, as indicated by CB-N and CB-C, respectively. The single-letter abbreviations for amino acids are used (see Chapter 2). cAMP-PK = cyclic AMP-dependent protein kinase CAM-MPK = Ca +/calmodulin-dependent multiprotein kinase PhK = phosphorylase kinase GSK = glycogen synthase kinase CK = casein kinase NIO-PK = A novel protein kinase. [Reproduced with permission from P. Cohen, Protein phosphorylation and hormone action. Proc. R. Soc. Lonrf. (Biol.) 234, 115(1988).]... 

Regulation of glycogen phosphorylase in muscle is accomplished by many of the same enzymes that control glycogen synthesis. Phosphorylase kinase converts the dimeric phosphorylase from the inactive to the active form by Mg + and ATP-dependent phosphorylation of two identical serine residues. The principal enzyme that removes this phosphate may be protein phosphatase-1 (phosphorylase phosphatase). 

Phosphorylation also can modify an enzyme s sensi-tivity to allosteric effectors. Phosphorylation of glycogen phosphorylase reduces its sensitivity to the allosteric activator adenosine monophosphate (AMP). Thus, a covalent modification triggered by an extracellular signal can override the influence of intracellular allosteric regulators. In other cases, variations in the concentrations of intracellular effectors can modify the response to the covalent modification, depending on the metabolic state of affairs in the cell. 

Fig. 7.9 Comparison of the allosteric regulation of gycogen phosphorylase with covalent regulation by phosphorylation dephosphorylation. The role of protein phosphatases in regulation was realized for the first time more than 50 years ago, as a cellular enzyme activity that converts the active a form of glycogen phosphorylase to an inactive b form.23...

Phosphorylation is another very common way of regulating enzymes, especially in signalling cascades. It requires ATP. A frequently quoted example is glycogen phosphorylase, an enzyme that phosphorylates glycogen, and is itself most active when phosphorylated. Phosphorylation of glycogen phosphorylase is reversible and controlled by the phosphorylase kinase and a phosphatase, (kinases add phosphate groups to proteins, phosphatases remove them). The phosphorylase kinase is itself regulated by phosphorylation (Fig. 6.19). 

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