Lecture 29 - Glycogen Breakdown and Synthesis

Molecular Structure and Physiological Roles of Glycogen

• Fundamental Definition and Distribution:

  • Glycogen is a large, highly branched homopolymer of glucose residues that serves as the primary carbohydrate storage form in animals.
  • It is present in all cell types and tissues across the human body, but the quantitatively largest stores reside in the liver and skeletal muscle.

• Tissue-Specific Physiological Functions:

  • Liver Glycogen Stores:
  • The liver maintains glycogen reserves that can be mobilized to buffer and maintain normal blood glucose concentrations.
  • Glucose released by hepatic glycogenolysis is exported into the systemic circulation to sustain energy demands of glucose-dependent peripheral tissues, principally the brain and erythrocytes (red blood cells).
  • Skeletal Muscle Glycogen Stores:
  • Muscle glycogen stores are dedicated entirely to local energy needs.
  • Mobilization occurs during muscle contraction and intensive exertion to fuel the glycolytic cascade and generate adenosine triphosphate ($\text{ATP}$) within muscle cells; it is not exported to the general circulation.

• Architecture of the Glycogen Storage Particle:

  • Central Core: The central anchor of the spherical granule is glycogenin, a specialized self-glucosylating protein that initiates glycogen assembly.
  • Linear Backbones: Glucose residues along linear chains are linked covalently by $\alpha\text{-}1,4\text{-glycosidic}$ bonds.
  • Branch Architecture: Branch points occur via $\alpha\text{-}1,6\text{-glycosidic}$ bonds approximately once every $10$ residues (ranging between $8$ and $12$ residues along each chain).
  • Exposed Periphery: The branched concentric layers terminate in numerous nonreducing ends exposed across the surface of the particle.

• Significance of High Branching:
  • Rapid Mobilization: The heavily branched architecture contrasts sharply with a single continuous linear strand (comparable to a pom-pom versus a tightly wound ball of yarn). Because degradative enzymes attack glycogen strictly from its nonreducing ends, the presence of many branching terminals allows simultaneous enzymatic cleavage by many phosphorylase molecules, ensuring rapid release of glucose when energy or blood sugar demands spike.
  • Enhanced Solubility: Branching prevents the aggregation and precipitation that would otherwise occur with long, unbranched glucan chains (such as amylose).

Glycogen Breakdown (Glycogenolysis): Enzymatic Machinery and Remodeling

• Enzymatic Requirements:

  • Complete degradation of glycogen to metabolizable intermediates requires four coordinated enzymes:
  1. Glycogen phosphorylase
  2. Glucosyltransferase (transferring activity of debranching enzyme)
  3. $\alpha\text{-}1,6\text{-Glucosidase}$ (hydrolytic activity of debranching enzyme)
  4. Phosphoglucomutase

• Phosphorolysis by Glycogen Phosphorylase:

  • Reaction Catalyzed: Glycogen phosphorylase removes single glucosyl residues sequentially from the nonreducing ends of the glycogen particle using inorganic phosphate ($\text{HPO}_4^{2-}$ or $\text{P}_i$) rather than water:     $\text{Glycogen}_{(n\text{ residues})} + \text{HPO}_4^{2-} \rightleftharpoons \text{Glucose 1-phosphate} + \text{Glycogen}_{(n - 1\text{ residues})}$
  • Thermodynamic and Bioenergetic Advantage: Phosphorolysis preserves the energy of the glycosidic bond in the form of a phosphate ester (glucose 1-phosphate). This spares the cell an $\text{ATP}$ molecule that would otherwise be required to phosphorylate free glucose via hexokinase upon entering glycolysis.
  • Substrate Specificity and Spatial Limitation: Phosphorylase is strictly specific for cleaving linear $\alpha\text{-}1,4\text{-glycosidic}$ bonds. Due to steric constraints in its catalytic cleft, cleavage stops four glucose residues away from an $\alpha\text{-}1,6$ branch point, leaving a steric block known as a "limit dextrin."

• Remodeling by the Debranching Enzyme System:
  • Transferase Activity: Glucosyltransferase cleaves a block of three glucosyl residues (a trisaccharide) from the four-residue limit branch and transfers it to the nonreducing end of an adjacent linear chain, extending that chain via an $\alpha\text{-}1,4\text{-linkage}$. This transfer unmasks the single glucose residue remaining at the branch point.
  • $\alpha\text{-}1,6\text{-Glucosidase}$ Activity: The exposed single glucose residue connected directly via an $\alpha\text{-}1,6\text{-linkage}$ is cleaved hydrolytically using a molecule of $\text{H}_2\text{O}$:     $\text{Glycogen}_{(n\text{ residues})} + \text{H}_2\text{O} \xrightarrow{\alpha\text{-}1,6\text{-Glucosidase}} \text{Glucose} + \text{Glycogen}_{(n - 1\text{ residues})}$
  • Products of Degradation:
  • Approximately $90\%$ of all glucosyl residues within glycogen are cleaved via phosphorolysis and released as glucose 1-phosphate.
  • Approximately $10\%$ of residues (those situated precisely at the $\alpha\text{-}1,6$ branch points) are cleaved via hydrolysis and released as free, unphosphorylated glucose.

• Isomerization by Phosphoglucomutase:
  • Conversion: Phosphoglucomutase catalyzes the reversible conversion of glucose 1-phosphate to glucose 6-phosphate.
  • Enzyme Active Site: The catalytic mechanism employs a phosphorylated serine residue (phosphoserine) at the active site.
  • Stepwise Mechanism:
  1. The phosphoenzyme donates its phosphoryl group to the C-6 hydroxyl of glucose 1-phosphate to generate an intermediate: glucose 1,6-bisphosphate.
  2. The enzyme abstracts the original phosphoryl group from the C-1 position of glucose 1,6-bisphosphate.
  3. Glucose 6-phosphate is released, and the serine residue is regenerated in its phosphorylated state for subsequent catalytic cycles.

Tissue-Specific Fates of Glucose 6-Phosphate

• Utilization in Skeletal Muscle:

  • Skeletal muscle lacks the enzyme glucose 6-phosphatase.
  • Because glucose 6-phosphate is charged and cannot cross the plasma membrane, it remains trapped inside the muscle fiber.
  • Muscle glucose 6-phosphate feeds directly into the glycolytic pathway to produce pyruvate, lactate, and $\text{ATP}$ to support mechanical work.
  • The small fraction of free glucose released in muscle by $\alpha\text{-}1,6\text{-glucosidase}$ is promptly phosphorylated to glucose 6-phosphate by hexokinase using $\text{ATP}$, entering glycolysis.

• Utilization and Glucose Export in the Liver:

  • The liver expresses glucose 6-phosphatase, allowing it to produce free glucose for systemic secretion and maintenance of homeostatic blood glucose levels.
  • Endoplasmic Reticulum Transport Complex:
  • The active site of glucose 6-phosphatase resides on the lumenal face of the endoplasmic reticulum ($\text{ER}$) membrane.
  • Transporter T1: Shuttles cytoplasmic glucose 6-phosphate into the lumen of the $\text{ER}$.
  • Catalytic Hydrolysis: The membrane-bound glucose 6-phosphatase hydrolyzes glucose 6-phosphate into inorganic phosphate ($\text{P}_i$) and free glucose:       $\text{Glucose 6-phosphate} + \text{H}_2\text{O} \rightarrow \text{Glucose} + \text{P}_i$
  • Transporters T2 and T3: Transporter T2 exports $\text{P}_i$ back to the cytoplasm, while Transporter T3 exports free glucose back to the cytoplasm.
  • Once in the cytoplasm, free glucose is exported through plasma membrane glucose transporters (GLUT) into the bloodstream.

Regulation of Glycogen Phosphorylase Isoforms

• Structural Dynamics and Dual Regulatory Control:
  • Glycogen phosphorylase is a homodimeric enzyme regulated by two distinct mechanisms:
  1. Reversible Covalent Modification: Phosphorylation/dephosphorylation of a specific serine residue.
  2. Allosteric Effectors: Reversible binding of small-molecule metabolites reflecting cellular energy status or substrate availability.
  • Conformational States:
  • Phosphorylase $a$: The phosphorylated form, which predominantly favors the catalytically active, open Relaxed ($\text{R}$) state.
  • Phosphorylase $b$: The unphosphorylated form, which predominantly favors the less active or inactive Tense ($\text{T}$) state due to active-site occlusion.

• Liver Phosphorylase Isoform Control:
  • Primary Objective: Maintenance of stable blood glucose levels.
  • Default State: The liver enzyme is normally in the phosphorylated $a$ form and locked in the active $\text{R}$ state (default is ON). The liver is primed to continually supply glucose to the bloodstream.
  • Allosteric Inactivation by Glucose:
  • Free glucose binds to allosteric sites on phosphorylase $a$.
  • Glucose acts as a negative allosteric effector, stabilizing the inactive $\text{T}$ state.
  • When blood glucose is adequate, liver glycogen breakdown is promptly repressed, and the $\text{T}$ state conformation exposes the regulatory phosphoserine residues to dephosphorylation by protein phosphatases.

• Muscle Phosphorylase Isoform Control:
  • Primary Objective: Rapid on-demand generation of $\text{ATP}$ to support mechanical contraction.
  • Default State: The muscle enzyme is normally in the unphosphorylated $b$ form and locked in the inactive $\text{T}$ state (default is OFF).
  • Allosteric Regulation via Energy Charge:
  • Positive Allosteric Effector ($\text{AMP}$): Elevated adenosine monophosphate ($\text{AMP}$) indicates energy depletion ($\text{low energy charge}$). Two molecules of $\text{AMP}$ bind to allosteric nucleotide-binding sites on phosphorylase $b$, stabilizing the active $\text{R}$ state even without phosphorylation.
  • Negative Allosteric Effectors ($\text{ATP}$ and Glucose 6-Phosphate): High concentrations of $\text{ATP}$ compete with $\text{AMP}$ for nucleotide-binding sites, while glucose 6-phosphate binds to an adjacent regulatory pocket; both act as negative allosteric effectors that preserve the inactive $\text{T}$ state.

Hormonal Activation and Termination Cascades in Glycogen Breakdown

• Hormonal Stimuli:

  • Glucagon: Secreted by pancreatic $\alpha\text{-cells}$ in response to low serum glucose; acts on hepatic tissue to mobilize glucose for systemic export.
  • Epinephrine (Adrenaline): Secreted by the adrenal medulla during stress, exercise, or acute "fight-or-flight" situations; acts primarily on skeletal muscle (and to a lesser degree on liver) to trigger massive, rapid glycogenolysis.

• The $7\text{-Transmembrane}$ ($7\text{TM}$) G-Protein Signal Transduction Cascade:

  1. Hormone binding to a surface $7\text{TM}$ receptor induces a conformational change on the intracellular face of the receptor.
  2. The receptor catalyzes guanosine diphosphate ($\text{GDP}$) to guanosine triphosphate ($\text{GTP}$) exchange on the $\alpha\text{-subunit}$ of the heterotrimeric G protein ($\text{G}_{\alpha\text{s}}$).
  3. Activated $\text{G}_{\alpha}\text{-GTP}$ dissociates from the $\beta\gamma\text{-dimer}$ and binds to and activates adenylate cyclase.
  4. Active adenylate cyclase converts $\text{ATP}$ into cyclic adenosine monophosphate ($\text{cAMP}$), the intracellular second messenger.
  5. $\text{cAMP}$ binds allosterically to the regulatory subunits of Protein Kinase A ($\text{PKA}$), releasing active catalytic subunits.
  6. Active $\text{PKA}$ phosphorylates phosphorylase kinase, converting it to its active state.
  7. Phosphorylase kinase phosphorylates glycogen phosphorylase $b$, converting it into active glycogen phosphorylase $a$.
  8. Active phosphorylase $a$ drives high-velocity phosphorolytic degradation of glycogen granules.

• Signal Termination Mechanisms:
  • Intrinsic GTPase Activity: The intrinsic GTPase activity of the $\text{G}_\alpha$ subunit hydrolyzes bound $\text{GTP}$ to $\text{GDP}$, terminating adenylate cyclase activation and promoting reassociation of $\text{G}_\alpha$ with $\text{G}_{\beta\gamma}$.
  • Phosphodiesterase Cleavage: Cyclic nucleotide phosphodiesterase hydrolyzes $\text{cAMP}$ into $5'\text{-AMP}$, an intermediate that cannot bind or activate $\text{PKA}$.
  • Dephosphorylation by Protein Phosphatase 1 ($\text{PP1}$): Insulin signaling stimulates $\text{PP1}$, which removes the activating phosphoryl groups from both phosphorylase kinase and glycogen phosphorylase $a$, rapidly restoring them to their unphosphorylated, basal inactive states.

Glycogen Synthesis (Glycogenesis): Pathway and Priming

• Pathway Separation:

  • Glycogen synthesis does not proceed via the simple reversal of glycogen phosphorylase.
  • Utilizing dedicated, thermodynamically distinct synthetic pathways prevents futile cycling and enables precise, reciprocal hormonal control.

• Synthesis of Activated Glucose (UDP-Glucose):

  • The activated donor of glucosyl moieties in glycogenesis is uridine diphosphate glucose ($\text{UDP-glucose}$).
  • Pyrophosphorylase Reaction: UDP-glucose pyrophosphorylase condenses glucose 1-phosphate with uridine triphosphate ($\text{UTP}$):     $\text{Glucose 1-phosphate} + \text{UTP} \rightleftharpoons \text{UDP-glucose} + \text{PP}_i$
  • Thermodynamic Coupling via Pyrophosphate Hydrolysis:
  • The reaction is freely reversible under standard conditions.
  • In vivo, inorganic pyrophosphatase immediately and irreversibly hydrolyzes inorganic pyrophosphate ($\text{PP}_i$) into two orthophosphate molecules ($2\,\text{P}_i$):       $\text{PP}_i + \text{H}_2\text{O} \rightarrow 2\,\text{P}_i$
  • Net reaction drive:       $\text{Glucose 1-phosphate} + \text{UTP} + \text{H}_2\text{O} \rightarrow \text{UDP-glucose} + 2\,\text{P}_i$
  • Rapid clearance of $\text{PP}_i$ pulls the pyrophosphorylase equilibrium forward, rendering the generation of $\text{UDP-glucose}$ practically irreversible.

• The Priming Function of Glycogenin:

  • Requirement for a Primer: Glycogen synthase is incapable of initiating glucan polymer synthesis de novo from individual glucose molecules; it strictly requires an existing oligosaccharide chain of at least four residues.
  • Glycogenin Properties:
  • Glycogenin is a specialized core protein arranged as a homodimer of two identical subunits.
  • Each subunit exhibits glucosyltransferase activity, transferring a glucose unit from $\text{UDP-glucose}$ to the hydroxyl group of a specific tyrosine residue on the opposing subunit.
  • Through iterative autocatalysis, each glycogenin subunit synthesizes an initial primer oligosaccharide consisting of $10\text{--}20$ glucose molecules linked by $\alpha\text{-}1,4\text{-bonds}$.
  • Glycogen synthase subsequently binds to this glycogenin-bound primer to carry out bulk chain elongation.

• Chain Extension by Glycogen Synthase:

  • Catalytic Function: Glycogen synthase transfers a glucosyl residue from $\text{UDP-glucose}$ to the C-4 hydroxyl group at the nonreducing end of a growing glycogen polymer.
  • Linkage Formed: The enzyme creates exclusively $\alpha\text{-}1,4\text{-glycosidic}$ linkages and releases $\text{UDP}$.

• Branch Formation by Glycogen Branching Enzyme:

  • Enzyme Identity: Amylo-$(1 \rightarrow 4) \rightarrow (1 \rightarrow 6)\text{-transglucosidase}$.
  • Mechanism of Branching:
  • The branching enzyme cleaves an $\alpha\text{-}1,4\text{-glycosidic}$ bond within an elongating chain that is at least $11$ residues long.
  • It takes a terminal block composed of approximately seven glucose residues and transfers it to the C-6 hydroxyl group of a more interior glucose residue on the same or an adjacent chain.
  • This creates a new $\alpha\text{-}1,6\text{-linkage}$, forming a branch point.
  • Consequences: Branching establishes a new nonreducing terminus, which allows both the newly created branch and the parent chain to be simultaneously extended by glycogen synthase.

Bioenergetics and Stoichiometric Efficiency of Glycogen Storage

• Stepwise Enzymatic Reactions of Glycogen Synthesis:

  1. $\text{Glucose} + \text{ATP} \xrightarrow{\text{Hexokinase}} \text{Glucose 6-phosphate} + \text{ADP}$
  2. $\text{Glucose 6-phosphate} \xrightarrow{\text{Phosphoglucomutase}} \text{Glucose 1-phosphate}$
  3. $\text{Glucose 1-phosphate} + \text{UTP} \xrightarrow{\text{UDP-glucose pyrophosphorylase}} \text{UDP-glucose} + \text{PP}_i$
  4. $\text{PP}_i + \text{H}_2\text{O} \xrightarrow{\text{Inorganic pyrophosphatase}} 2\,\text{P}_i$
  5. $\text{UDP-glucose} + \text{Glycogen}_n \xrightarrow{\text{Glycogen synthase}} \text{Glycogen}_{n+1} + \text{UDP}$
  6. $\text{UDP} + \text{ATP} \xrightarrow{\text{Nucleoside diphosphate kinase}} \text{UTP} + \text{ADP}$

• Overall Net Equation for Incorporation of Dietary Glucose:$\text{Glucose} + 2\,\text{ATP} + \text{Glycogen}_n + \text{H}_2\text{O} \rightarrow \text{Glycogen}_{n+1} + 2\,\text{ADP} + 2\,\text{P}_i$

• Energy Balance and Storage Efficiency:

  • Storage Cost: Storing one molecule of circulating glucose into glycogen costs the cell exactly two high-energy phosphate bonds ($2\,\text{ATP}$ equivalents: one $\text{ATP}$ for glucose phosphorylation by hexokinase, and one $\text{ATP}$ to regenerate $\text{UTP}$ from $\text{UDP}$).
  • Oxidation Yield: Complete aerobic oxidation of a single glucose residue derived via glycogen phosphorolysis yields $31\,\text{ATP}$.
  • Storage Efficiency Calculation:$\text{Storage Cost Fraction} = \frac{2}{31} \approx 6.5\%$$\text{Storage Efficiency} = 100\% - 6.5\% = 93.5\%$
  • Storing glucose as a polymeric glycogen reserve is biologically efficient, conserving approximately $93.5\%$ of the potential energetic yield of the carbohydrate fuel.

Reciprocal and Coordinated Regulation of Glycogen Metabolism

• Core Regulatory Logic:

  • Covalent phosphorylation coordinates the opposing branches of glycogen metabolism in opposite directions:
  • Phosphorylation: Turns breakdown ON (activates glycogen phosphorylase) and turns synthesis OFF (inactivates glycogen synthase).
  • Dephosphorylation: Turns breakdown OFF (inactivates glycogen phosphorylase) and turns synthesis ON (activates glycogen synthase).

• Regulation of Glycogen Synthase:

  • Covalent Modification:
  • Glycogen Synthase $a$: Unphosphorylated and active.
  • Glycogen Synthase $b$: Phosphorylated and inactive (or substantially less active).
  • Kinases Involved: Protein Kinase A ($\text{PKA}$) and glycogen synthase kinase ($\text{GSK}$) phosphorylate synthase $a$ to produce inactive synthase $b$.
  • Allosteric Modulation:
  • Glucose 6-phosphate acts as a positive allosteric effector for glycogen synthase.
  • High glucose 6-phosphate concentrations stabilize the active $\text{R}$ state of phosphorylated glycogen synthase $b$, stimulating glycogenesis when precursor availability is abundant.

• Integrated Hormone-Driven Regulation:

  • Fasting or Exertion (Glucagon / Epinephrine Dominance):
  • Epinephrine (in muscle and liver) and Glucagon (in liver) stimulate $7\text{TM}$ receptors, driving $\text{cAMP}$ generation and activating $\text{PKA}$.
  • $\text{PKA}$ phosphorylates phosphorylase kinase, which phosphorylates glycogen phosphorylase $b$ to $a$, stimulating glycogen breakdown.
  • Concurrently, $\text{PKA}$ and $\text{GSK}$ phosphorylate glycogen synthase $a$ to $b$, shutting off glycogen synthesis.
  • This reciprocal regulation prevents a futile cycle of simultaneous synthesis and breakdown.

• Fed State (Insulin Dominance):
  • High blood glucose levels trigger insulin secretion from pancreatic $\beta\text{-cells}$.
  • Insulin signaling activates Protein Phosphatase 1 ($\text{PP1}$).
  • Active $\text{PP1}$ dephosphorylates phosphorylase kinase and glycogen phosphorylase $a$, switching off glycogenolysis.
  • Concurrently, $\text{PP1}$ dephosphorylates glycogen synthase $b$, converting it into active glycogen synthase $a$, which accelerates glycogen synthesis.

• Summary Comparison of Key Enzymes in Glycogen Metabolism:
  • Glycogen Phosphorylase:
  • Active state: Phosphorylated ($a$ form, $\text{R}$ state).
  • Inactive state: Dephosphorylated ($b$ form, $\text{T}$ state).
  • Liver allostery: Glucose acts as a negative effector.
  • Muscle allostery: $\text{AMP}$ acts as a positive effector; $\text{ATP}$ and glucose 6-phosphate act as negative effectors.
  • Glycogen Synthase:
  • Active state: Dephosphorylated ($a$ form, $\text{R}$ state).
  • Inactive state: Phosphorylated ($b$ form, $\text{T}$ state).
  • Allostery: Glucose 6-phosphate acts as a positive allosteric effector for the $b$ form.
  • Hormonal Switch:
  • Epinephrine and Glucagon: Stimulate degradation, repress synthesis via $\text{cAMP}$ and $\text{PKA}$.
  • Insulin: Stimulates synthesis, represses degradation via $\text{PP1}$.