GPBB

Glycogen phosphorylase (EC 2.4.1.1, GP) is one of the phosphorylase enzymes that catalyzes the hydrolysis of glycogen to generate glucose-1-phosphate and shortened glycogen molecule. It catalyzes the rate-limiting step in the degradation of glycogen. Glycogen phosphorylase is also studied as a model protein regulated by both reversible phosphorylation and allosteric effects. Studies have found that mammals have liver, muscle and brain isoforms of glycogen phosphorylase. Muscle glycogen phosphorylase is present to degrade glycogen to forms of energy by means of glycolysis during muscle contractions, and liver glycogen is present to regulate the blood glucose levels within the blood.

Isoforms

There are three isoforms of glycogen phosphorylase encoded by 3 genes PYGL, PYGM and PYGB, and designated Liver, Muscle and Brain after the tissues in which they are expressed. There is high sequence identity (~97%) between human and respective rodent isoforms, and 80% identity between the liver isoform and the muscle or brain isoforms. The latter share 83% identity. Glycogen phosphorylase is a dimer composed of monomers of 846 (PYGL), 841 (PYGM) or 862 (PYGB) residues with pyridoxal phosphate, an essential cofactor, in the center of each monomer covalently linked to a lysine residue. All 3 isoforms of glycogen phosphorylase are regulated allosterically by binding of several metabolite effectors and by reversible phosphorylation of serine-14. Phosphorylase was the first enzyme shown to be regulated by covalent phosphorylation. Its regulation by acetylation was discovered recently. The phosphorylated and dephosphorylated forms are designated, respectively, GPa and GPb.

Structure

Glycogen phosphorylase consists of two domains. The N-terminal domain contains several regulatory sites. One is serine-14 which is modified by phosphorylation and lies within a region (residues 5–22) that adopts an ordered or disordered conformation across the subunit interface depending on the phosphorylation state. Another is the allosteric “AMP activator site” that binds AMP and several other phosphoryl ligands including G6P, ATP and fructose 1-phosphate. This domain also has the glycogen storage allosteric site and components of the binding sites for purines and glucose as well as the residues at the subunit interface that are required for propagating the allosteric effects. The C-terminal domain contains the cofactor binding site and together with residues from the N-terminal domain contains the catalytic site in a deep crevice, which is sheltered by a moveable gate comprising residues of the N-terminal domain (280-285 residues). The purine inhibitor site is at the entrance to the catalytic site.

The dimer of glycogen phosphorylase has two faces: one with access to the catalytic site that would be in contact with glycogen and a regulatory face containing the N-terminal tail and the AMP-activator site exposed to the cytoplasm. The catalytic site is at the dimer interface and has the pyridoxal cofactor covalently bound. Access of substrate is guarded by a gate that has an open position in the R-state and a closed position in the T-state that is stabilized by glucose. The purine inhibitor is formed by contacts between the gate and the C-terminal domain.

Mechanism

Glycogen phosphorylase breaks up glycogen into glucose subunits. It can act only on linear chains of glycogen (α1-4 glycosidic linkage). Although the reaction is reversible in solution, within the cell the enzyme only works in the forward direction because the concentration of inorganic phosphate is much higher than that of glucose-1-phosphate. Its work will immediately come to a halt four residues away from α1-6 branch (which are exceedingly common in glycogen). In these situations, a debranching enzyme is necessary, which will straighten out the chain in that area. In addition, the enzyme transferase shifts a block of 3 glucosyl residues from the outer branch to the other end, and then a α1-6 glucosidase enzyme is required to break the remaining (single glucose) α1-6 residue that remains in the new linear chain. After all this is done, glycogen phosphorylase can continue.

Figure 1. Action of glycogen phosphorylase on glycogen.

Regulation

Changes in liver glycogen storage during the diurnal cycle are associated with changes in the activities of glycogen phosphorylase (GP) and glycogen synthetase (GS) which regulated in a reciprocal manner. Reciprocal changes in activities of GP and GS are in part mediated by a glycogen targeted binding protein of protein phosphatase-1 (PP1c) that positively regulates the activity of GS and has an allosteric inhibitor site for the activated GP. Accordingly, activation of GP determines not only the rate of degradation of glycogen but also inhibition of activation of GS. Regulation of GP is therefore a major upstream event in the control of glycogen turnover by both extracellular and intracellular signals.

Application

The inhibition of glycogen phosphorylase has been proposed as one method for treating type 2 diabetes. Since glucose production in the liver has been shown to increase in type 2 diabetes patients, inhibiting the release of glucose from the liver’s glycogen’s supplies appears to be a valid approach. The cloning of the human liver glycogen phosphorylase (HLGP) revealed a new allosteric binding site near the subunit interface that is not present in the rabbit muscle glycogen phosphorylase (RMGP) normally used in studies. This site was not sensitive to the same inhibitors as those at the AMP allosteric site, and most success has been had synthesizing new inhibitors that mimic the structure of glucose, since glucose-6-phosphate is a known inhibitor of HLGP and stabilizes the less active T-state.

Reference

1. Agius L. Role of glycogen phosphorylase in liver glycogen metabolism. Molecular Aspects of Medicine, 2015, 46:34-45.