Glycogen phosphorylase B (PDB ID: 8GPB) from Oryctolagus cuniculus

Created by: Brianna Kelly

              Glycogen phosphorylase b (PBD ID: 8GPB), from Oryctolagus cuniculus, serves an important biological role as the inactive form of the enzyme, glycogen phosphorylase, which when active breaks glycogen into glucose-1-phosphate in the muscle cells (1).  Glucose-1-phosphate is then converted to glycogen-6-phosphate which undergoes glycolysis to provide energy for muscle contraction.  Overall, glycogen phosphorylase b in muscle cells starts the process of converting glycogen into energy in the form of ATP.  Crystallization, using x-ray diffraction, of the entire protein from the muscles of Oryctolagus cuniculus allows for the study of the structural basis for the protein’s function.   Glycogen phosphorylase b is a single subunit structure, which allows exposed residues to bind ligands to promote its transformation to its active state.  The primary structure of glycogen phosphorylase b consists of 842 amino acids.  Secondary structures contained in the protein include 27 beta sheets, 42 alpha helices, random coils, 8 3/10 helices, and one pi helix. The globular protein’s structure consists of a primarily beta sheet core surrounded by layers of alpha helices on the exterior.  Glycogen phosphorylase b has a molecular weight of 97158.22 daltons and an isoelectric point of 6.77 (2).  

            Glycogen phosphorylase b has two ligands of importance to its function. Adenosine Monophosphate is of particular interest in research as it binds to provide regulation by allosteric control (1).  When bound to Tyr-155, adenosine monophosphate allows glycogen phosphorylase b to change from its inactive T state to its active R state.  This allows the enzyme to perform its function of cleaving the glycosidic linkage and breaking down glycogen without being phosphorylated (3).   The activation of the enzyme via allosteric control contrasts its usual activation via phosphorylation.  Pyridoxal-5’-phosphate is another ligand of interest for glycogen phosphorylase b.  This ligand is also a cofactor and binds to Lys-680 via a Schiff Base linkage (3).  Pyridoxal-5’-phosphate assists the reaction of the breakdown of glycogen into glucose by binding adjacent to the active site.  Without this prosthetic group, glycogen phosphorylase b would not be able to perform its function of binding the phosphate in order to use it in the phosphorolysis of glycogen (3). 

            As mentioned with the ligands, glycogen phosphorylase b has many functionally important residues.  Many of the important residues are found at the catalytic or binding sites.  In order for glycogen phosphorylase b to be phosphorylated, a phosphate must bind to Ser-14 (1).  This binding exhibits the covalent control of the enzyme to change its conformation from inactive T state glycogen phosphorylase b to active glycogen phosphorylase a.  This is the common mechanism for activation of glycogen phosphorylase b in order to break down glycogen.  The protein also exhibits important hydrogen bonding at this binding site as the phosphate hydrogen bonds with the nearby Arg-16 to stabilize the binding of the phosphate to Ser-14 (1).  Arg-16 also further hydrogen bonds with Ser-14 to complete the stable binding site for phosphorylation of glycogen phosphorylase b (4).  Other residues important for this binding site include Asp-283 and Arg-569.  Asp-283, a negatively charged residue, is found in the active site when glycogen phosphorylase b is in the T state.  Upon activation to the R state, Asp-283 is displaced and replaced by Arg-569 in the active site.  Asp-283 creates an unfavorable environment in the active site for binding phosphate due to its negative charge.  By replacing it with positively charged Arg-569, the switch provides for a higher affinity phosphate-binding site (4).  Conversely, Tyr-155 serves as the catalytic site for the binding of adenosine monophosphate.  As discussed previously, this provides the enzyme with allosteric control.  When adenosine monophosphate binds it transforms glycogen phosphorylase b into its active R state without changing the enzyme completely.Cys-108 and Cys-142 are also functionally important residues for glycogen phosphorylase b.  These residues control the association of ligands and the activity of the enzyme.  These residues are normally free and open; however, when they are blocked they cause the enzyme to become inactive.  Finally, Lys-680 is important for binding pyridoxal 5’-phosphate.  The amino group on Lys-680 allows for the reaction with pyridoxal-5'-phosphate to form the necessary Schiff base linkage which holds the prosthetic group in place (3).  Without this binding site, phosphorylation of glycogen phosphorylase b would not be possible and the enzyme would remain inactive.  

            Glycogen phosphorylase b has two different states, its inactive T state and active R state.  Upon activation by allosteric control, glycogen phosphorylase b goes from its T state to its R state.  This change helps to enhance the binding site for adenosine monophosphate (1).   This is due to the increased affinity for adenosine monophosphate that occurs upon the transition from the T to R state (1). Once glycogen phosphorylase b is activated by adenosine monophosphate it forms tetramers in its R state.  Glycogen phosphorylase b can also undergo a conformation change to the tetrameric glycogen phosphorylase a.  The phosphorylation of glycogen phosphorylase b causes this change specifically in the tertiary and quaternary structures.  The catalytic site is originally inaccessible in the T state of glycogen phosphorylase b due to restrictions created by the loop of residues 282 to 286 (4).  The catalytic site, measured to be 15 angstroms below the surface of the protein, can only be accessed at this point via the narrow tunnel created by the loop (4,5).  However, upon activation the loop shifts position and displaces thus exposing the catalytic site and allowing for the binding of pyridoxal 5’-phosphate.  The displacement of the loop occurs due to the transition to the active R state which causes the tower helices, residues 262-276, to shift (5).  While in the T state the tower helices are associated in an antiparallel formation with an angle of tilt about -20° (1).  These are shifted to have a angle of tilt about -70° when glycogen phosphorylase b is activated to the R state.

The structure of glycogen phosphorylase b is similar to subunit A of maltodextrin phosphorylase (PDB ID: 1L5V), found in Escherichia coli.  The Dali server and psi-Blast allowed for numerical comparisons of the structures and sequences for these proteins.  The Dali server compares the intermolecular distances of the proteins and then produces a Z-score.  The Z-score for structural similarities is 48.7, indicating that the tertiary structures are moderately similar considering the magnitude of 48.7 compared to the ideal Z-score being greater than 2 (6).   The psi-Blast compares the primary structure of proteins and produces E values to indicate total sequence homology.  The E-value for subunit A of maltodextrin phosphorylase is very low, either zero or nearly zero, indicating that the sequences are very similar compared to the ideal E-value being less than 0.05 (7). 

Subunit A of maltodextrin phosphorylase and glycogen phosphorylase b exhibit the most structural similarity at their catalytic sites (8).  However, maltodextrin phosphorylase does not require activation by control, as the catalytic site remains open on the protein making it only found in its active state in Escherichia coli.  Due to the differences between control of the enzymes, most of the differences occur around these regions (9).  Specifically, most differences occur on the exterior portions of the proteins in which there is a deletion of certain loops on the surface.  These loops found in glycogen phosphorylase b serve the purpose of blocking the catalytic site.  However, by removing these loops from maltodextrin phosphorylase, the enzyme avoids having to undergo a conformation change.  This deletion also makes maltodextrin phosphorylase a more compact protein.  Overall, both enzymes have very similar secondary structures especially in the internal beta strand core (9).  Glycogen phosphorylase b and maltodextrin phosphorylase also differ in their number of subunits. Maltodextrin phosphorylase has two subunits while glycogen phosphorylase b has only one subunit.  Glycogen phosphorylase b specifically differs in its orientation of its subunit, as the positioning of its T and R states do not match either of the two subunits for maltodextrin phosphorylase (9).  Finally, the major sequence differences are found at the glycogen binding site as maltodextrin phosphorylase has a very low affinity for glycogen.  This is due to a deletion of many of the residues found at the site which result in a loss of the hydrophobic interactions and hydrogen bonding found in glycogen phosphorylase b.  Therefore, instead maltodextrin phosphorylase has a high affinity for linear oligosaccharides, such as maltodextrin, rather than the branched polysaccharide, glycogen (8).

Glycogen phosphorylase b serves an important role in allowing for the breakdown of glycogen in order to release ATP in muscle cells, specifically found in the organism Oryctolagus cuniculus.  Glycogen phosphorylase b is of particular interest due to its activation via allosteric control as well as via phosphorylation (4).  Allosterically activating glycogen phosphorylase b allows for the release of high amounts of energy when an organism such as Oryctolagus cuniculus participates in exercise and thus depletes its stores of ATP faster.