15.3: Glycogenolysis and its Regulation by Glucagon and Epinephrine Signaling

Search Fundamentals of Biochemistry

Learning Goals (ChatGPT o3-mini)

1. Understand the Overall Process of Glycogenolysis:

   • Describe the stepwise breakdown of glycogen into glucose-1-phosphate and free glucose, and explain how the end products differ between liver (glucose release into circulation) and skeletal muscle (glucose-6-phosphate for glycolysis).

2. Identify the Key Enzymes in Glycogenolysis:

   • Explain the roles of glycogen phosphorylase and glycogen debranching enzyme in catalyzing the sequential breakdown of glycogen, including how glycogen phosphorylase acts on alpha 1→4 linkages and the debranching enzyme resolves alpha 1→6 branches.

3. Analyze the Mechanism of Glycogen Phosphorylase:

   • Detail the reaction mechanism of glycogen phosphorylase, including the function of the pyridoxal phosphate (PLP) cofactor (attached via a Schiff base with a lysine residue) and how inorganic phosphate is utilized to cleave glycosidic bonds.

4. Examine the Function of Glycogen Debranching Enzyme:

   • Outline the dual catalytic activities of the glycogen debranching enzyme (glycosyl transferase and glucosidase) that enable the enzyme to process branch points and allow continuous glycogen breakdown.

5. Discuss Hormonal Regulation of Glycogenolysis:

   • Compare and contrast the roles of glucagon and epinephrine in activating glycogenolysis, including their effects on liver and skeletal muscle.
   • Explain how glucagon signaling downregulates glycogen synthesis and promotes glycogen breakdown in the liver, while epinephrine activates glycogenolysis in both liver and muscle tissues.

6. Understand the Glucagon Signaling Cascade:

   • Describe the glucagon receptor as a G-protein-coupled receptor and outline the signaling cascade that leads to cAMP production, activation of protein kinase A (PKA), and the subsequent phosphorylation of key regulatory proteins.

7. Examine the Regulation of Glycogen Synthase and Phosphorylase:

   • Detail how PKA-mediated phosphorylation inactivates glycogen synthase (and its associated protein phosphatase PP1) while activating phosphorylase kinase, which in turn phosphorylates and activates glycogen phosphorylase.

8. Evaluate Allosteric and Isozyme Regulation in Glycogenolysis:

   • Discuss the allosteric regulation of glycogen phosphorylase by metabolites such as glucose (in liver) and AMP (in skeletal muscle) that shift the enzyme between active (R) and inactive (T) states.
   • Compare the tissue-specific differences between liver and muscle isozymes of glycogen phosphorylase in terms of their regulatory properties.

9. Connect Glycogenolysis to Overall Glucose Metabolism:

   • Integrate how glucose-1-phosphate, produced from glycogen breakdown, is converted to glucose-6-phosphate and directed into glycolysis, gluconeogenesis, or other metabolic pathways.
   • Relate these processes to the body’s energy homeostasis and the role of the liver in maintaining blood glucose levels.

10. Understand the Clinical Implications:

    • Explain how deficiencies in glycogen phosphorylase (as in McArdle's Disease) affect muscle metabolism, leading to exercise intolerance, muscle cramping, and potential secondary complications (e.g., myoglobinuria and kidney damage).

These learning goals are designed to build a detailed understanding of the enzymatic mechanisms, regulatory pathways, and physiological significance of glycogenolysis and its hormonal control in maintaining metabolic balance.

In the previous section, you learned that glucagon signaling down-regulates glycogen synthesis. Now, let's look at glycogen breakdown, called glycogenolysis, and its control by two hormones, glucagon and epinephrine. Only two enzymes are required for the breakdown of glycogen: the glycogen phosphorylase enzyme and the glycogen debranching enzyme.

Glycogenolysis: An Overview

Two key enzymes are required for the stepwise catabolism of glycogen: glycogen phosphorylase and glycogen debranching enzyme. In the liver, the ultimate end product is glucose-1-phosphate, which is dephosphorylated in the liver to enable the export of free glucose into the circulation. In contrast, in the respiring skeletal muscle, it is converted to glucose-6-phosphate for glycolysis. Glycogenolysis is also activated by the hormones glucagon and epinephrine.

Glycogen Phosphorylase

Glycogen phosphorylase (GP) catalyzes the release of glucose 1-phosphate from the alpha 1→ 4 non-reducing glycogen ends. Figure \(\PageIndex{1}\) shows an overview of this reaction.

Figure \(\PageIndex{1}\): Overview of Glycogen Phosphorylase Reaction. Images from Ascherer730 and Michal Sobkowski

Glycogen phosphorylase is a homodimer with two active sites. It also requires a cofactor, pyridoxal phosphate (PLP) to be functional (Figure \(\PageIndex{2}\)). The PLP is derived from Vitamin B6. You may have previously heard that low B vitamin levels are associated with lethargy or a lack of energy. We will continue to see that the B vitamins provide essential cofactors for enzymes involved in the production of ATP. Thus, lacking B vitamins means you are not efficiently producing ATP. The PLP cofactor of GP is attached covalently to the enzyme through a Schiff-base linkage with a Lysine (K) residue.

Figure \(\PageIndex{2}\): Pyridoxyl Phosphate Cofactor Associated with Glycogen Phosphorylase Image modified from Ascherer730

The reaction mechanism of glycogen phosphorylase is detailed in Figure \(\PageIndex{3}\). When glycogen phosphorylase binds with glycogen, a free inorganic phosphate anion is positioned by the PLP and the enzyme active site in proximity with the anomeric carbon position of the non-reducing end residue of the glycogen molecule. The oxygen in the glycosidic bond attacks the partially charged hydrogen associated with the phosphate ion, leading to the cleavage of the glycosidic bond. The cleaved glycogen chain leaves the active site, and one of the phosphate oxygens attacks the carbocation intermediate created during the cleavage. This results in the release of the terminal glucose residue as glucose 1-phosphate

Figure \(\PageIndex {3}\): Glycogen Phosphorylase Reaction Mechanism. Image from Ascherer730

Glycogen Debranching Enzyme

Glycogen phosphorylase cannot cleave the alpha 1 → 6 linkages. It also cannot cleave α,1 → 4 linkages that are within four residues of an α,1→ 6 linkage (the glycogen chain will no longer fit into the active site of the enzyme). The Glycogen Debranching Enzyme (GDE) has two catalytic activities that enable it to deal with this problem. The first catalytic activity is a Glycosyl Transferase (GT) activity. In this process, the three remaining alpha 1 → 4 extended units on the branch site (colored in green) are clipped off of the branch site and attached to a straight chain of alpha 1 → 4 extended glucose residues. The second part of the reaction requires glucosidase (GC) activity, which mediates the hydrolysis of the α,1 → 6 glycosidic bond and releases free glucose in the process. Glycogen phosphorylase can then resume the breakdown of the remaining alpha 1 → 4 chain. Figure \(\PageIndex{4}\) shows an overview of glycogen breakdown.

Figure \(\PageIndex {4}\): Biological Activity of the Glycogen Debranching Enzyme. Image modified from XiangSong

Dephosphorylation of Glucose 1-Phosphate

Following the activation of glycogenolysis, the liver cell has now released large quantities of glucose 1-phosphate from glycogen and a smaller amount of free glucose from the clipped branch residues. The free glucose can be transported to the bloodstream straight away, but the glucose 1-phosphate must be dephosphorylated before release (Figure \(\PageIndex{5}\)).

Figure \(\PageIndex{5}\): Process of Glucose Dephosphorylation in Liver Cells

The dephosphorylation of glucose only occurs in liver cells, as this is the primary location for regulating blood glucose levels. Free glucose can exit the cell, whereas phosphorylated forms are trapped inside. Figure \(\PageIndex{6}\) outlines the process of glucose dephosphorylation in the liver. To mediate the dephosphorylation of glucose, glucose 6-phosphate is transported from the cytoplasm into the lumen of the endoplasmic reticulum (ER) through transporter 1 (T1). The glucose-6-phosphatase (G-6-Pase) then cleaves the phosphate from the substrate, releasing inorganic phosphate (P) and glucose (represented by the red molecule). The inorganic phosphate is then transported back into the cytoplasm through transporter 2 (T2), and glucose is transported through Transporter 3 (T3). Free glucose is then transported back into the bloodstream through a glucose (GLUT) transporter in the plasma membrane.

Figure \(\PageIndex{6}\): Dephosphorylation of Glucose 6-phosphate in Liver Cells.

Hormonal Control of Glycogen Breakdown

In the previous sections, we’ve discussed insulin signaling and the process of building glycogen (glycogenesis) in detail. Now, look at the other side of the homeostatic balance, which begins with glucagon signaling. During hypoglycemia (or low blood glucose levels), pancreatic alpha (α) cells release the hormone peptide, glucagon, which stimulates gluconeogenesis (the formation of glucose) and glycogenolysis (the breakdown of glycogen) in the liver, resulting in the release of glucose to the plasma, and the raising of blood glucose levels, as shown in Figure \(\PageIndex{7}\).

Figure \(\PageIndex{7}\): Summary of Glucagon Signaling During Low Blood Glucose Levels. Image from Hædersdal, S., et al (2018) Mayo Clinic Proceedings 93(2):217-239

Let’s review a few terms before we begin. In the previous section, we were introduced to glycogenesis, the process of synthesizing glycogen. We saw that this pathway was activated during insulin signaling. In glucagon signaling, this pathway is inhibited and the opposite pathway, glycogenolysis (glycogen breakdown) is activated. Glucagon signaling in the liver also downregulates glycolysis (the utilization of glucose for energy production), as the liver attempts to utilize glucose to maintain blood glucose levels. It doesn’t utilize it for its own energy needs during this time. Instead, liver cells can use lipids to generate ATP, and in fact, glucagon signaling increases lipolysis or the breakdown of lipids. Finally, glucagon also up-regulates gluconeogenesis, or the generation of glucose from non-sugar metabolites. In a later section, we will address the mechanisms of glycolysis and gluconeogenesis regulation. Here, we will only take a cursory look at these pathways and focus more on the process of glycogenolysis.

Overview of Glucagon Signaling

Glucagon signaling begins when the hormone binds with its receptor on liver cells, as shown in Figure \(\PageIndex{8}\). Glucagon receptors are not as widespread within the body as insulin receptors. Since the purpose of this hormone is to cause the release of glucose back into the bloodstream, this process is highly controlled, and only the liver can deliver glucose back into the bloodstream to maintain homeostasis. Thus, other target tissues, such as skeletal muscle, do not require these receptors to be expressed and are therefore not sensitive to glucagon signaling.

Figure \(\PageIndex{8}\): Overview of Glucagon Signaling Cascade

The glucagon receptor is a G-protein-coupled receptor, also called a 7TM receptor (as it contains seven transmembrane helices that span the plasma membrane). This family of receptors is widespread throughout the body and is responsible for many of the pharmaceutical mechanisms of action seen in our treatment of different disease conditions. With this pathway, once glucagon binds to the receptor, the receptor moves laterally in the plasma membrane. It binds with a G-protein, an intracellular peripheral protein. The G-protein consists of three major domains: the alpha, beta, and gamma domains. The alpha domain is capable of binding to the GDP/GTP cofactor. When the G-protein is inactive, all three subunits stay together, and the alpha subunit remains inactive and bound to GDP.

When the G-protein associates with an activated receptor, the alpha subunit exchanges GTP for the bound GDP cofactor, and the gamma and beta subunits dissociate into the cytoplasm. The activated alpha subunit moves laterally on the periphery of the plasma membrane until it contacts the adenylyl cyclase enzyme (also called adenylate cyclase). This activates the adenylyl cyclase, which converts ATP into cyclic adenosine monophosphate (cAMP). cAMP production is an amplification step within this pathway. That means that more cAMP is produced than G-proteins are activated.

After some time, a G-protein hydrolase causes the hydrolysis of the GTP to GDP and inactivates the G-protein. At this point, the G-protein associates with the gamma and beta subunits, reforming its inactive state. Another glucagon signaling event will be required to reactivate the process. The cyclic AMP produced in the process serves as a second messenger and activates a myriad of downstream targets. We will focus on two of the major targets.

The first is Protein Kinase A, which becomes activated upon binding with cAMP. The second target is a cAMP Response Element-Binding Protein (CREB). The CREB protein is also activated when bound to the cAMP molecule. This causes the CREB protein to translocate from the cytosol into the mitochondria and the nucleus. In both locations, the activated CREB binds to specific response element sequences in the DNA and activates the transcription of genes involved in gluconeogenesis. These genes and their encoded proteins have been discussed in more detail in Chapter 14. What is important to note is that glucagon signaling in the liver results in the upregulation of glucose production de novo from non-carbohydrate precursors. This is NOT a favored pathway in the body. It is energetically expensive for the liver to manufacture glucose. In fact, the cost of ATP is more expensive than can be produced from the newly formed molecule. However, organs like the brain can only utilize free glucose as an energy source. Thus, the liver will use this energy deficit to build glucose for use by the brain and other cellular targets.

Glucagon signaling also leads to the downregulation of glycolysis, which we will cover in more depth in Section 15.4, as well as glycogenesis. It also leads to an increase in glycogenolysis, or the breakdown of glycogen. Let's examine the regulation of both of these processes in more detail.

Regulation of Glycogenesis 

Since glycogen synthase (GS) is the primary enzyme required for glycogenesis, it is also the primary target for regulating this pathway. Recall that GS is active in the dephosphorylated state. Thus, PKA down-regulates the activity of this enzyme through the phosphorylation of GS, as shown in Figure \(\PageIndex{9}\). Phosphorylation of GS causes it to shift into its inactive conformation, inhibiting glycogenesis.

Figure \(\PageIndex{9}\): Inactivation of Glycogen Synthase through Phosphorylation. When bound to cAMP, protein kinase A (PKA) phosphorylates and inactivates the glycogen synthase enzyme. The activity of protein phosphorylase 1 (PP1) is required to dephosphorylate GS and restore its activity. Image modified from Yan, A., et al (2016) In J Biol Sci 12(12):1544-1554 and Servier Medical Art

Additionally, activated PKA phosphorylates the protein phosphatase 1 (PP1) enzyme, resulting in the inactivation of the phosphatase. PP1 normally dephosphorylates GS, helping to retain the active conformation of GS. Thus, phosphorylation of PP1 by PKA helps to maintain the GS in the phosphorylated, inactive state. The inhibition of PP1 is quite complicated, as shown in Figure \(\PageIndex{10}\). PP1 contains a regulatory domain and a catalytic domain. Normally, the regulatory domain of PP1 binds to glycogen, keeping the molecule close to the location where GS is present. Thus, when GS is near its substrate, it can bind with PP1 and be dephosphorylated into its active state. This is more efficient than diffusing in the cell and trying to find the PP1 randomly. When PKA phosphorylates the regulatory domain of PP1, it dissociates from the catalytic domain, causing the catalytic domain to float away from the glycogen molecule. This makes PP1 less efficient at dephosphorylating GS because it is more difficult for the molecules to interact randomly. Thus, PP1 is less active. PKA further reduces this activity by phosphorylating an allosteric inhibitor (I) of PP1. In the phosphorylated state, the inhibitor can bind to PP1, inactivating the phosphatase. Both phosphorylation events need to be reversed to regain full PP1 activity.

Figure \(\PageIndex{10}\): Inactivation of Phosphorylase 1 (PP1) by Protein Kinase A (PKA). PKA phosphorylates PP1 causing it to dissociate from glycogen and become less active. PKA also phosphorylates an allosteric inhibitor (I) of PP1 which increases its binding affinity for PP1. The phosphorylated inhibitor maintains PP1 in an inactive conformation. Image Modified from Yan, A., et al (2016) In J Biol Sci 12(12):1544-1554 and Servier Medical Art

In summary, glucagon signaling in the liver downregulates glycogenesis by activating PKA. PKA phosphorylates GS directly, inactivating the enzyme, and maintains it in the inactive state by also inhibiting the PP1 responsible for dephosphorylating GS.

Activation of Glycogenolysis

In addition to phosphorylating GS and PP1 during the inactivation of glycogenesis, PKA also phosphorylates the enzyme Phosphorylase Kinase, which is upstream of glycogen phosphorylase, the primary enzyme involved in glycogen breakdown. As its name implies, phosphorylase kinase is a protein kinase that phosphorylates the enzyme to activate it. Figure \(\PageIndex{11}\) offers a first view of the phosphorylation cascade required for glycogen phosphorylase activation.

Figure \(\PageIndex{11}\): Activation of Glycogen Phosphorylase by Protein Kinase A (PKA) Signaling. PKA upregulates the activity of Phosphorylase Kinase through direct phosphorylation. The activated kinase enzyme phosphorylates its downstream target, Glycogen Phosphorylase (GP). In the phosphorylated state, GP is more active. Image modified from Yan, A., et al (2016) In J Biol Sci 12(12):1544-1554 and Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

The phosphorylase kinase enzyme is a complex enzyme that is a tetramer of a tetrameric complex, αβγδ, so the full holoenzyme has an (αβγδ)₄ structure. It is large, with a molecular weight of around 1.3x10⁶. As expected, it is highly regulated in multiple ways, including phosphorylation by PKA, ADP (an allosteric effector), divalent cations like Ca²⁺, and pH. The α and β are regulatory subunits that affect activity through their phosphorylation. The δ is calmodulin, a calcium-binding protein we discussed in Chapter 12.7, and its binding of calcium affects the holoenzyme activity. The γ subunit has kinase activity, an N-terminal catalytic domain, and a C-terminal calmodulin-binding domain.  This is primarily regulated by phosphorylation through the PKA pathway, as shown in Figure 15.3.5.

"PHK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (αβγδ)₄, and a total molecular weight. wt of 1.3×10⁶ Da. Activity is regulated by cyclic AMP-dependent protein kinase phosphorylation, autophosphorylation, allosteric effectors (e.g., ADP), metal ion concentration (Ca²⁺ and Mg²⁺), proteolysis, and pH (Pickett-Gies and Walsh, 1986). The α and β subunits are regulatory and are the targets for control by phosphorylation. The δ subunit is essentially identical to calmodulin and confers Ca²⁺ sensitivity. The 386 amino acid γ subunit is the catalytic subunit, which comprises an N-terminal kinase domain (residues 1–298) and a regulatory calmodulin-binding domain (residues 299–386)."

Calcium, an allosteric regulator, may be present within cellular targets due to nerve impulse firing, muscle contraction, or hormone signaling. Calcium in the cell generally indicates a high cellular energy demand at that time, indicating that energy production is needed. Thus, calcium binding to the phosphorylase kinase is a positive effector of the enzyme, upregulating its activity. The enzyme's maximal activity is achieved through combined phosphorylation and calcium binding. Thus, phosphorylase kinase can exist in 4 different activity states as shown in linked equilibria in Figure \(\PageIndex{12}\).

Figure \(\PageIndex{12}\): Activation States of Phosphorylase Kinase (PK). In the left-hand diagram, PK is in the inactive state, with the kinase-containing alpha domains shown in red. The upper diagram shows the activation of PK through phosphorylation by Protein Kinase A during hormone signaling. This leads to a partially active enzyme. Similarly, calcium binding, shown in the lower diagram, also results in a partially active enzyme. Calcium plays a particularly important role in the activation of this enzyme in skeletal muscle. The process of muscle contraction causes the release of high levels of calcium into the cytoplasm. Thus, the presence of calcium within the cytoplasm of muscle cells indicates high energy demand, as the muscle is being called into action. This activates PK and stimulates the breakdown of glycogen within muscle tissue, helping to meet energy demands. Maximal activity is obtained with both calcium binding and phosphorylation.

This diagram should be quite familiar by now. It is yet another example of a tetrameric enzyme (where the "enzyme" composition is αβγδ) existing in two major states: an inactive T state and an active R state. The interconversion is regulated by allosteric effectors (Ca2+) and is post-translationally phosphorylated.

A mechanism for the phosphorylation of a Ser in a substrate target protein (i.e, glycogen phosphorylase) by the catalytic domain of the γ subunit of phosphorylase kinase is shown in Figure \(\PageIndex{13}\).

Figure \(\PageIndex{13}\): A mechanism for the phosphorylation of a Ser in a substrate target protein (i.e, glycogen phosphorylase) by the catalytic domain of the γ subunit of phosphorylase kinase. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/35/. Creative Commons Attribution 4.0 International (CC BY 4.0) License.

The * in the mechanism denotes the serine of the target protein.

Figure \(\PageIndex{14}\) below shows an interactive iCn3D model of a truncated form of the rabbit phosphorylase kinase gamma subunit dimer bound to a peptide substrate complex (2PHK).

Figure \(\PageIndex{14}\): Rabbit phosphorylase kinase gamma subunit dimer bound to a peptide substrate complex (2PHK). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...3wHapyHWEy7qi9 

The phosphorylase kinase dimer is shown (gray and brown subunits).  A non-hydrolyzable ATP analog (adenylyl imidodiphosphate, AMPPNP) in each subunit is shown as CPK-colored sticks. The backbone of two identical peptide substrates (blue and cyan) with the sequence RQMSFRL (similar to the target sequence in glycogen phosphorylase and an ideal peptide substrate) is shown as a backbone trace, with the central Ser, which gets phosphorylated, represented as CPK-colored spheres. Active site residues are shown as CPK-colored sticks and labeled in each subunit.  

We have been primarily discussing the regulation of glycogenolysis in the liver. However, in considering the activity of PK and its reactivity with Ca²⁺ ions, we should also consider the activation of glycogenolysis in skeletal muscle. Notably, glycogenolysis in liver tissue and skeletal muscle has many differences. First, the G-protein-coupled pathway is activated by different hormones. Liver tissue is responsive to Glucagon stimulation and stimulation through the Epinephrine hormone signaling pathway. Glycogenolysis in skeletal muscle tissue, on the other hand, is only activated by the Epinephrine signaling pathway, but not by glucagon. This is because the liver is the primary organ responsible for regulating blood glucose levels. Thus, due to low blood glucose levels, pancreatic signaling primarily targets glycogenolysis within the liver tissue. Both systems are responsive to epinephrine, which is described in more detail below.

Epinephrine Signaling

Epinephrine is a small amino acid-derived hormone (can you guess the amino acid?? Yes, it is tyrosine!!), as shown in Figure \(\PageIndex{15}\). It is also known as adrenaline, which is secreted by the adrenal glands located above the kidneys during the fight-or-flight response. It is also secreted during heavy or sustained exercise. Epinephrine has pleiotropic effects in the body, including the activation of glycogenolysis in the liver and skeletal muscles. Epinephrine also promotes fat breakdown in adipose tissue, which releases this energy reserve into the bloodstream for utilization by muscle tissue. It also causes the relaxation of smooth muscles in the lungs and respiratory tract, allowing for improved oxygen absorption. Cardiac contractility is also increased to increase blood flow to skeletal muscles. This supports the generation of ATP from glucose and fatty acids for sustained muscle utilization. It also reduces blood flow to the skin and causes the contraction of smooth muscles in the ski,n causing goosebumps.

Figure \(\PageIndex{15}\): Structure of Epinephrine

Within the liver and skeletal muscle, the epinephrine signaling pathway overlaps with the glucagon signaling pathway, which is also present in the liver. The epinephrine receptor is also a G protein-coupled receptor related to the glucagon receptor. However, it is specific for epinephrine and cannot bind with glucagon. It does activate the same G-protein pathway leading to Protein Kinase A activation, as shown in Figure \(\PageIndex{16}\). The body is very efficient at reusing machinery in different parts of the body; in this case, it does so under different regulatory parameters.

Figure \(\PageIndex{16}\): Similarities of Glucagon and Epinephrine Signaling Pathways

The glycogen phosphorylase enzymes are also encoded by different genes within the liver and skeletal muscle. These are known as isozymes. Recall that isozymes have the same biological function, but since they are expressed from different genes, they have different enzyme kinetics and they are regulated in different and unique ways within each tissue.

The liver and skeletal muscle forms of Glycogen Phosphorylase share approximately 90% sequence identity. Both isozymes can exist in two major conformations, the a-form and the b-form. The protein adopts the a-form when it is phosphorylated at Ser 12 by phosphorylase kinase, as shown in Figure \(\PageIndex{17}\). The Glycogen Phosphorylase enzyme can also be in two different states: the relaxed, flexible state, which is the active form of the enzyme, and the tense or rigid state, which is inactive. When the protein is in the a-conformation, it favors the relaxed and active state of the protein. Therefore, the phosphorylation of Glycogen Phosphorylase leads to an increase in the enzyme's activity. This is depicted in the following diagram.

Figure \(\PageIndex{17}\): Enzymatic States of Liver and Skeletal Muscle Glycogen Phosphorylase. Both isozymes of Glycogen Phosphorylase are responsive to phosphorylation by phosphorylase kinase. Phosphorylation of Glycogen Phosphorylase causes it to shift from the b-form to the a-form of the protein. The a-form of the protein favors the relaxed, active state of the protein, whereas the b-form of the protein favors the tense and inactive state. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

Another way to think about these four different enzyme states is that they exist in a dynamic equilibrium, with the population of each state determined by the phosphorylation state AND the presence of allosteric inhibitors and activators, which we will explore below. In Figure 17 above, the R state is positioned at the top, and the T state is positioned at the bottom. The vertical arrows indicate the equilibrium between just those two states. The thickness of the arrows indicates the preferred direction of the reversible reaction. In the absence of phosphorylation (left-hand vertical states), the equilibrium favors the T or inactive form. When phosphorylated (right-hand vertical states) on Ser12 by the enzyme phosphorylase kinase, the R or active form is favored. The horizontal equilibria show the phosphorylation of Ser12 by phosphorylase kinase (top, shown reversibly, but the enzyme is not acting physiologically to remove phosphate) and dephosphorylation of Ser 12 by the enzyme protein phosphatase 1 (which acts physiologically only as a phosphatase).

Different tissue-specific allosteric effectors also regulate the different isozymes of the Glycogen Phosphorylase enzyme. Within the liver, glucose acts as a negative regulator of Glycogen Phosphorylase, which is logical, given the role of this pathway in liver tissue, which is to promote the release of glucose into the bloodstream. The presence of free glucose in the cytoplasm of the liver would indicate either the fed state, when blood glucose levels are high, or that high levels of glycogenolysis have released substantial glucose. Within liver tissue, the presence of free glucose causes the a-form of Glycogen Phosphorylase to shift to the Tense state, reducing enzyme activity. This is shown in Figure \(\PageIndex{18}\).

Figure \(\PageIndex{18}\): Regulation of Liver Glycogen Phosphorylase by Free Glucose. When the liver isoform of Glycogen Phosphorylase is in the a-form (phosphorylated), it can be shifted into the Tense state in the presence of high levels of free glucose. This blocks the glycogen binding site of the enzyme, essentially serving as a competitive inhibitor. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

Skeletal muscle glycogen phosphorylase (or Myophosphorylase, as it is sometimes called) is more responsive to allosteric effectors that indicate the energy state of the cell. This makes sense, as the primary purpose of glycogen breakdown in muscle tissue is to meet the energy demands of the muscle tissue. Thus, the energy housed in glucose will be used to produce ATP within these cells. The presence of either glucose 6-phosphate or ATP within skeletal muscle indicates high energy levels. Thus, glycogen breakdown will be inhibited. On the other hand, the presence of AMP indicates a low-energy state and serves as an activator of Glycogen Phosphorylase. This is shown in Figure \(\PageIndex{19}\) to Figure \(\PageIndex{21}\)below.

Figure \(\PageIndex{19}\): Regulation of Skeletal Muscle Glycogen Phosphorylase by Glucose 6-phosphate. The presence of high levels of Glucose 6-phosphate negatively regulates Muscle Glycogen Phosphorylase. Regardless of the form (a or b) the enzyme is shifted into the Tense state. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

Again, note that the bold red arrows point downward, showing that the addition of glucose-6-phosphate favors the T state (inactive) even if glycogen phosphorylase has been phosphorylated.

Figure \(\PageIndex{20}\): Regulation of Skeletal Muscle Glycogen Phosphorylase by ATP. In the presence of high levels of ATP, GP is converted to the Tense state, showing the inhibition of the enzyme in the presence of high levels of energy. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

Again, note that the bold red arrows point downward, indicating that when ATP levels are high, the T state (inactive) is favored, even if glycogen phosphorylase has been phosphorylated. Again, under conditions of a high energy state (as reflected by high ATP), there is no need to cleave glycogen to enter glycolysis.

Figure \(\PageIndex{21}\): Regulation of Skeletal Muscle Glycogen Phosphorylase by AMP. In the presence of low-energy indicators, such as AMP, the glycogen phosphorylase enzyme is activated, even in the absence of phosphorylation. Thus, when AMP is bound, the b-form of glycogen phosphorylase is converted into the relaxed and active state. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

In contrast, note that the bold green arrows point upward, showing that when the AMP levels are relatively high, the R state is active. Higher levels of AMP reflect a need to activate glycogen breakdown to increase ATP production.

A mechanism for the phosphorolysis of Glc_n+1 to Glc_n and glucose-1-phosphate is shown in Figure \(\PageIndex{22}\). Note the unusual presence of a molecule of pyridoxal phosphate covalently attached through a Schiff base linkage to Lys 568 (rabbit phosphorylase).

Figure \(\PageIndex{22}\): A mechanism for the phosphorolysis of Glc_n+1 to Glc_n and glucose-1-phosphate. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/205/.  Creative Commons Attribution 4.0 International (CC BY 4.0) License

PLP, which is covalently attached through a Schiff base, functions in this enzyme as a general acid and base and not as a cofactor that facilitates covalent bond cleavage in amino acid substrates that are covalently attached to it (Chapter 6.8: Cofactors and Catalysis - A Little Help From My Friends).

It is essential to note that the reaction is a phosphorolysis, not a hydrolysis, which would result in the release of free glucose. This glucose would then be more readily available to leave the cell, making it less accessible for cellular energy needs and less available for glycolysis.

Figure \(\PageIndex{23}\) below shows an interactive iCn3D model of a dimer of rabbit glycogen phosphorylase (1GDB).

Figure \(\PageIndex{23}\): Dimer of rabbit glycogen phosphorylase (1GDB). (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...s1LosR5TJLjru9

The subunits in the dimeric form are shown in different colors. PLP is shown in spacefill. Active site residues are shown in both subunits as CPK-colored sticks and labeled.

Let's look at a monomer (from the tetramer) of 2 nonphosphorylated states of glycogen phosphorylase. Since they are both unphosphorylated, they both represent the b state. One (2GPB) has glucose bound, so it represents the inactive (T state). The other (3E3N) has AMP bound, so it represents the active (the R state). Figure \(\PageIndex{24}\) shows the conformation differences between the monomeric states

Figure \(\PageIndex{24}\): Conformational differences between the monomeric unphosphorylated b state of glucose-bound GP (T state, inactive) and AMP-bound GP (R state, active)

Now, let's examine the conformations of two different GPs activated by distinct means. In one, the nonphosphorylated form of GP (the b state and inactive T state) binds AMP, an allosteric activator (pdb 8GPA), and converts to the active b state (unphosphorylated R state). Let's compare its active conformation to a form of GP activated by phosphorylation (the phosphorylated a state and active R state) to which just SO₄^2- is also bound. In the 1^st case, GP-b T state is driven to the active Gp-b R state by binding the allosteric activator AMP. In the second case, GP-a is already in the R active state since it is phosphorylated.  Figure \(\PageIndex{25}\) compares their conformations.

Figure \(\PageIndex{25}\): Comparison of the conformations of two active forms of GP - phosphorylase b bound to the allosteric activator AMP (R state) and phosphorylase a activated by phosphorylation of Ser 14.

The cyan monomer is glycogen phosphorylase b, which is not phosphorylated but is driven into the R state upon the binding of AMP (note that two AMP molecules are bound at the periphery). The dark blue monomer is glycogen phosphorylase A, which is phosphorylated at Ser14 (a different number in this crystal file, as shown in spacefill and labeled SEP-14), also in the R state. It also has two SO₄^2- bound, which help stabilize the state. Look carefully! The conformations are very similar, in contrast to those for the T and R states shown in Figure 24.

Figure \(\PageIndex{26}\) below shows an interactive iCn3D model of unphosphorylated (b state) rabbit glycogen phosphorylase with bound glucose (inactive T state, 2GPB).

Figure \(\PageIndex{26}\): Unphosphorylated (b state) rabbit glycogen phosphorylase with bound glucose (inactive T state, 2GPB). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...eXQciAPczmQpLA

Only 1 monomer of the tetramer is shown. PLP and glucose are shown in spacefill, CPK colors, and labeled. Key active site residues are shown as colored sticks and labeled.

Figure \(\PageIndex{27}\) below shows an interactive iCn3D model of unphosphorylated (b state) rabbit glycogen phosphorylase with bound AMP (active R state, 3E3N).

Figure \(\PageIndex{27}\): Unphosphorylated (b state) rabbit glycogen phosphorylase with bound AMP (active R state, 3E3N). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...WDZJc1CcpFqEG7

Note the different locations for the binding site of the allosteric activator AMP compared to the inhibitor glucose in the previous model.

McArdle's Disease

McArdle's Disease, also referred to as myophosphorylase deficiency or type V glycogen storage disease, is a recessive inherited disorder characterized by an inability to metabolize glycogen due to the absence of a functional myophosphorylase (PYGM). In Figure \(\PageIndex{28}\) shown below, the normal functional pathway is shown in blue on the left, while the mutant pathway is shown on the right in red. Patients with this disease lack sufficient glucose-1-phosphate (G1P) monomers for glycolysis and the hexosamine biosynthetic pathway (HBP). This results in lower ATP and, consequently, lower muscle contraction and lower post-translational modifications by O-GlcNAcylation compared to normal conditions. This is especially pronounced during extended or heavy workouts, where people with McArdle’s Disease will sustain painful cramping of their muscle tissue during workouts, can have dark red/brown urine, and can easily tire during activity. Some patients also experience a second-wind phenomenon during workouts, as the body shifts from carbohydrates to lipids as its primary energy source. The urine's dark red/brown color happens if muscle tissue is damaged during the workout. The damaged muscle releases the protein myoglobin into the bloodstream. This is filtered out by the kidneys and excreted in the urine, causing the color change. The severe, uncontrolled disease can cause life-threatening kidney problems.

Figure \(\PageIndex{28}\): McArdle's Disease. The left-hand side (blue) represents the normal pathway, whereas the right-hand side (red) notes the deficiency of muscle glycogen phosphorylase. Image from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

Figure \(\PageIndex{29}\) presents an overview of glucose metabolism in skeletal muscle. Both glucose-1-phosphate (G1P) released from the intracellular glycogen stores by glycogen phosphorylase (GP), as well as the glucose introduced into the cell through glucose transporters (GLUT), are converted to glucose-6-phosphate (G6P) by phosphoglucomutase (PGM) and hexokinase (HK), respectively. The G6P can be directed to different destinations. One is the pentose phosphate pathway, which is used for forming nucleic acid building blocks (ribose and deoxyribose). Another is in the formation of ATP. Here, G6P enters the glycolysis metabolic pathway. The glycolytic reactions culminate in the production of pyruvate and adenosine triphosphate (ATP). Pyruvate can be fermented to lactate by lactate dehydrogenase (LDH) catalysis during anaerobic muscle exercise.

On the other hand, pyruvate can be used to obtain ATP through full oxidation in the Krebs Cycle. In total, oxidative phosphorylation produces between 30-36 molecules of ATP (depending on the organism and tissue), six molecules of carbon dioxide (CO₂), and six molecules of water (H₂O) from 1 glucose molecule. Glycolysis alone only produces two net ATP molecules per glucose. Glucose, in addition to being the main fuel of the cell’s energy metabolism, is also used by the cellular machinery as a vitally important substrate for the production of key intermediaries of the hexosamine biosynthetic pathway (HBP) forming O-GlcNAc, β-linked N-acetylglucosamine. And finally, in times of plenty, glucose is utilized by glycogen synthase (GS) to form glycogen.

Figure \(\PageIndex{29}\): Summary of Glucose Metabolism in Skeletal Muscle. Image from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

Summary

This chapter provides an in-depth exploration of glycogenolysis—the enzymatic breakdown of glycogen—and its regulation by the hormones glucagon and epinephrine, highlighting key differences between liver and skeletal muscle metabolism.

Overview of Glycogenolysis:
The process of glycogenolysis is initiated by two key enzymes: glycogen phosphorylase and the glycogen debranching enzyme. Glycogen phosphorylase cleaves α(1→4) glycosidic bonds at the non-reducing ends of glycogen, releasing glucose 1-phosphate. In contrast, the debranching enzyme overcomes the limitations imposed by branch points (α(1→6) linkages) by transferring and hydrolyzing short glucan fragments, allowing for continued degradation of glycogen.

Mechanism of Glycogen Phosphorylase:
Glycogen phosphorylase is a homodimer that requires pyridoxal phosphate (PLP) as a cofactor, which is covalently linked to the enzyme via a lysine residue. The enzyme positions a free inorganic phosphate in its active site, facilitating the nucleophilic attack on the glycosidic bond, thereby releasing glucose 1-phosphate. This reaction is critical for generating substrates for further metabolic processes.

Hormonal Regulation and Tissue Specificity:
Hormonal signals tightly regulate glycogenolysis. In the liver, glucagon is released during hypoglycemia, triggering a signaling cascade via its G-protein-coupled receptor. This cascade leads to an increase in cyclic AMP (cAMP), activation of protein kinase A (PKA), and subsequent phosphorylation of key enzymes:

• Inactivation of Glycogen Synthase (GS): PKA phosphorylates GS, shifting it into an inactive state and preventing glycogen synthesis.
• Activation of Glycogen Phosphorylase: PKA also activates phosphorylase kinase, which in turn phosphorylates glycogen phosphorylase, converting it from the less active b form to the active a form. Additionally, calcium binding further enhances phosphorylase kinase activity, particularly in skeletal muscle.

Skeletal muscle, however, responds primarily to epinephrine rather than glucagon. Epinephrine also activates the cAMP/PKA pathway but, in muscle, glycogenolysis is regulated by allosteric effectors:

• Inhibition by ATP and Glucose 6-Phosphate: High levels indicate abundant energy and downregulate enzyme activity.
• Activation by AMP: High AMP levels signal low energy, promoting the active state of glycogen phosphorylase.

Conformational States and Allosteric Regulation:
Glycogen phosphorylase exists in dynamic equilibrium between inactive (T) and active (R) conformations. The enzyme's activity is modulated by phosphorylation and by binding of allosteric effectors. Structural models illustrate how binding of inhibitors (e.g., free glucose in the liver) or activators (e.g., AMP in muscle) shifts the equilibrium, thereby finely tuning the enzyme's catalytic performance.

Integration with Overall Glucose Metabolism:
The products of glycogenolysis serve different roles in liver and muscle tissues. In the liver, glucose 1-phosphate is eventually dephosphorylated to free glucose for release into the bloodstream, crucial for maintaining blood glucose levels and supporting brain function. In skeletal muscle, glucose 6-phosphate enters glycolysis to meet immediate energy demands.

Clinical Relevance:
The chapter concludes by highlighting the clinical implications of defective glycogenolysis. For instance, McArdle's Disease (myophosphorylase deficiency) disrupts muscle energy metabolism, leading to exercise intolerance, muscle cramping, and potential kidney complications due to myoglobinuria.

Overall, this chapter bridges the molecular mechanisms of enzyme action with hormonal regulation and systemic metabolic balance, offering a comprehensive understanding of how glycogen breakdown is finely controlled to meet the energy demands of the body.