Glycogen Phosphorylase B
Created by: Michael Sohn
Glycogen phosphorylase B (PDB ID= 8GPB) is a phosphorylase enzyme that catalyzes the rate-limiting step in the process of glycogenolysis in animals by releasing glucose-1-phosphate from the terminal α1,4-glycosidic bond. Glycogenolysis is the process by which glycogen is broken down into glucose-1-phosphate and glucose. (3) This process occurs in both muscle and liver cells in response to hormonal or neural signaling and plays a crucial role in regulation of blood glucose levels. Glycogen phosphorylase is researched as a model protein affected by reversible phosphorylation and allosteric effects. In addition, due to its involvement in the regulation of blood glucose levels, glycogen phosphorylase is being researched as a possible tool in curing or reducing the effects of type-2 diabetes. Inhibiting Glycogen phosphorylase B activity would reduce the amount of glucose in the blood and the subsequent consequences associated with type 2 diabetes. Mutations in various isoforms of Glycogen Phosphorylase B is linked to various diseases in brain, liver, and muscle tissue. (8)
The molecular weight of GPB is 97,158.22 Da, and the isoelectric point is at pH 6.77. (2) Glycogen Phosphorylase B is considered to be the inactive form in comparison to glycogen phosphorylase a. Glycogen Phosphorylase B is usually in the inactive T state under the presence of ATP and glucose-6-phosphate whereas glycogen phosphorylase a is usually in the active R state. This difference in activity is caused by the disordered residues located at Arg-10 to Glu-22 in Glycogen Phosphorylase B being organized into α helices in glycogen phosphorylase A. (5) Glycogen Phosphorylase B was also the first allosteric enzyme discovered and was originally studied in Oryctalagus Cuniculis, more commonly known as the rabbit. (7)
Glycogen Phosphorylase B is a homodimer of two identical subunits (842 residues, 97.44 kDa). Both subunits contain an active site, an allosteric effector site near the subunit surface, a regulatory phosphorylation site at Ser-14, and a glycogen-binding site which facilitates association to its substrate as well as serving in a regulatory role. (4) The catalytic site is relatively buried underneath the subunit surface (15Å). Access to the site is created through allosteric effects. The regulatory site at Ser-14 is a site of reversible phosphorylation located close to the subunit surface. (6) Another important residue is Arg-10. At the N terminus of this residue, one of the most important intersubunit interactions are present involving the helix alpha 2 and the cap’. The interactions present induce structural changes at the helix/cap interface that create the high-affinity AMP site. In addition, the tertiary and quaternary changes create greater affinity and four additional hydrogen bonds, stronger ionic interactions, and greater Van der Waal interactions to facilitate the AMP binding site. (3)
Glycogen phosphorylase is composed of 8 beta sheets (17%), 23 alpha helices (48%), and 17 random coils (35%). In addition, several tower helices are present. These components of secondary structure form the basis for the structure of the protein including the catalytic (active) site as well as other binding sites of the protein. Glycogen Phosphorylase B can exist in two states, R or T, depending on the presence of adenosine monophosphate (AMP). The allosteric site for AMP binding is also located close to the subunit interface. When AMP binds at this site, a corresponding change from the T to R state occurs, leading to small changes in tertiary structure and large changes in quaternary structure. The two tower helices of the two subunits rotate 50o relative to one another. This results in the subunits rotating 10o relative to one another and opens the previously blocked catalytic site at residues Asn-282 to Phe-286. The glycogen storage site is located at residues Pro-397 to Val-437. This site covalently binds glycogen chains 30 Å from the catalytic site and is most likely the area where the protein binds to glycogen granules before cleaving the terminal glucose molecules. (3) In addition, a pyridoxal-5’-phophate (PLP) is located at each catalytic site. PLP connects with basic residues of the protein (ex. Arg and Lys-680) and covalently forms a Schiff base. The Schiff base linkage holds the PLP molecule at the catalytic site and allows PLP to donate a proton from its phosphate group to an inorganic phosphate (pi) which is then deprotonated by an oxygen forming the α1,4-glycosidic linkage. Glycogen phosphorylase can only act on α1,4-glycosidic linkages as the 30 Å gap connecting the glycogen storage site to the active site provides enough space for the helix formed by the glycogen chain, accommodating 4 to 5 glucosyl residues, but not enough space for branches of more complex structures. (5) Other important bonds of glycogen phosphorylase occur at N6 of AMP to Lys-315, N6 of AMP and Cys-318, N1 of AMP and Gly-317, and Lys-315 and Glu-785. These locations are sites of hydrogen bonds that are crucial to the structure and function of the protein. In addition, other important residues include the side chains of Phe-285 and Tyr-613 as the side chains to these residues near the catalytic site tunnel bind inhibitors to glycogen phosphorylase. (3)
Glycogen Phosphorylase is regulated both by allosteric control and phosphorylation. Hormones including epinephrine, glucagon, and insulin aid in regulating glycogen phosphorylase using second messenger amplification systems involving G proteins. These hormones act through different phosphorylation cascades that eventually lead to the activiation of glycogen phosphorylase kinase and the subsequent phosphorylation of Glycogen Phosphorylase B at Ser-14, transforming it into the active glycogen phosphorylase a. (8) Glycogen Phosphorylase B can also be activated allosterically by AMP in muscle tissue. (6) An increase in AMP concentration (occurs during strenuous exercise) signals energy demand causing AMP to change the conformation of Glycogen Phosphorylase B from a tense to more relaxed form. The relaxed form has similar enzymatic properties to the phosphorylated enzyme. Activation through AMP is inhibited by high ATP concentration which causes the displacement of AMP from the nucleotide binding site which indicates sufficient energy storage. (10)
There are numerous methods available to find detailed information regarding the sequence and structure of a myriad of proteins. One such database is the Dali server. The Dali server compares tertiary structures of proteins and calculates the differences in intramolecular distances using the “sums-of-pairs method.” This results in a value called the z-score with a score higher than a 2 meaning the proteins have similar tertiary structure (folds). Another database available is the PSI-BLAST program. This program is used to find proteins of similar primary structure to the inquired protein. PSI-BLAST calculates an e-value which is measured by comparing the sequence of the queried protein to other proteins and assigning gaps, amino acids that exist in the subject protein but not the query protein. An e-value of less than 0.5 is indicative of high similarity between proteins. The comparison protein chosen was glycogen phosphorylase of the species homo sapiens or humans. The z score between these two proteins was 61.1 and the e value was 0.0. (1,9) Both of these values are significant and indicate high tertiary structure similarity (z-score) and high similarity in the sequences of the two proteins (e-value). Human glycogen phosphorylase shares one ligand between them, AMP. Glycogen phosphorylase in humans does not utilize pyridoxyl-5’-phosphate as an associated ligand as they do in rabbits. Instead, it utilizes two additional ligands not associated with rabbit glycogen phosphorylase, adenine and alpha-d-glucose. These two ligands function to perform in the same capacity as glycogen phosphorylase in rabbits and do not differ in any significant way. The two phosphorylases are otherwise, structurally very similar composed of 2 subunits and similar amounts of the same secondary, tertiary, and quaternary structure. However, human glycogen phosphorylase functions in a similar manner as rabbit glycogen phosphorylase with two states, active R and inactive T, as well as being regulated by phosphorylation and allosterically. The two phosphorylases are not known to utilized in any protein-drug complexes and do not have any associated metal ions.