Phosphorylation glycogen phosphorylase

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

As early as 1938, it was known that glycogen phosphorylase existed in two forms the less active phosphorylase b and the more active phosphorylase a. In 1956, Edwin Krebs and Edmond Eischer reported that a converting enzyme could convert phosphorylase b to phosphorylase a. Three years later, Krebs and Eischer demonstrated that the conversion of phosphorylase b to phosphorylase a involved covalent phosphorylation, as in Eigure 15.17. 

Johnson, L. N., 1992. Glycogen phosphorylase Control by phosphorylation and allosteric effectors. FASEB Journal 6 2274-2282. 

The principal enzymes controlling glycogen metabolism—glycogen phosphorylase and glycogen synthase— are regulated by allosteric mechanisms and covalent modifications due to reversible phosphorylation and... 

SIGNAL INTEGRATION by phosphorylase kinase. Phosphorylase kinase eventually phosphorylates and activates glycogen phosphorylase. Either (or both) phosphorylation and calcium signaling pathways converge at phosphorylase kinase. 

DG). InsP3, as we saw above, stimulates the release of Ca2+, sequestered in the ER, and this in turn activates numerous cellular processes through Ca2+-binding proteins, such as calmodulin. The membrane-associated DG activates protein kinase C to phosphorylate and activate other enzymes, such as glycogen phosphorylase. This step also requires Ca2+. 

Glycogen phosphorylase isoenzymes have been isolated from liver, brain and skeletal muscle. All forms are subject to covalent control with conversion of the inactive forms (GP-b) to the active forms (GP-a) by phosphorylation on specific serine residues. This phosphorylation step, mediated by the enzyme phosphorylase kinase, is initiated by glucagon stimulation of the hepatocyte. Indeed, the same cAMP cascade which inhibits glycogen synthesis simultaneously stimulates glycogenolysis, giving us an excellent example of reciprocal control. 

Muscle glycogen phosphorylase is one of the most well studied enzymes and was also one of the first enzymes discovered to be controlled by reversible phosphorylation (by E.G. Krebs and E. Fischer in 1956). Phosphorylase is also controlled allosterically by ATP, AMP, glucose and glucose-6-phosphate. Structurally, muscle glycogen phosphorylase is similar to its hepatic isoenzyme counterpart composed of identical subunits each with a molecular mass of approximately 110 kDa. To achieve full activity, the enzyme requires the binding of one molecule of pyridoxal phosphate, the active form of vitamin B6, to each subunit. 

A well-known example, indeed the first enzyme that was shown to be regulated by the phosphorylation/ dephosphorylation mechanism, is glycogen phosphorylase, which catalyses the breakdown of glycogen (Box 3.7). 

Covalent interconversion of enzymes is well established as a fundamental theme in metabolic regulation. The prototypic reversible interconverting systems include the sequence of phosphorylation/dephosphorylation steps in the activation of mammalian glycogen phosphorylase and pyruvate dehydrogenase as well as the nucleotidyla-tion/denucleotidylation using UTP and ATP in the bacterial glutamine synthetase cascade (see Fig. 1.). 

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. 

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... 

Fig. 2.12. Structural changes at the N-terminus of glycogen phosphorylase as a result of phosphorylation. a) R-form of the dimer of glycogen phosphorylase a. b) T form of the dimer of glycogen phosphorylase b. Phosphorylation at Serl3 near the N-terminus transforms the inactive glycogen phosphorylase b into the active glycogen phosphorylase a. The N-terminus rearranges significantly as a result of phosphorylation. In the inactive T-state the N-terminus interacts with the same subunit, while in the R-form it forms interactions with the other subunit. After Barford and Johnson (1991), with permission.

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.

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