Glycolysis Vocabulary

Announcements

• TCA quiz is scheduled for this coming Friday (11/22) during lecture. It will cover identical concepts to the glycolysis quiz, so make sure to review those principles.

• Exolife extra credit project offers an opportunity to add 1.25% to your total grade.

  • The journal entry should include a detailed description of a lifeform, its biome, and its biochemistry.

  • Lifeform: Elaborate on its physiology, behavior, and interactions within its indigenous population.

  • Biome: Describe the climate, temperature, and other relevant environmental factors.

  • Biochemistry: Base the lifeform's biochemistry on real-world systems, demonstrating your understanding of scientific principles through a fictional context.

  • Details for journal entry components:

  • Lifeform: Provide an in-depth description of the organism's physical attributes, behavioral patterns, and interactions with both its environment and other organisms. Focus on any unique adaptations and strategies it employs for survival.

  • Biome: Give a thorough account of the environmental conditions, including climate, temperature ranges, and the availability of essential resources. Explain how these factors directly influence the lifeform's adaptations and overall survival.

  • Biochemistry: Expound upon the biochemical processes that facilitate and sustain the lifeform's existence. Draw comparisons with real-world biochemical systems, emphasizing energy production, metabolic pathways, and any distinctive molecular adaptations.

  • The due date for this project is December 5th at 11:59 PM.

Glycolysis

• Glycolysis is the metabolic process involving the breakdown of glucose into pyruvate.

  • Detailed Definition: Glycolysis is a biochemical pathway that extracts energy from glucose by cleaving it into two molecules of pyruvate, each containing three carbon atoms. This process unfolds in the cytoplasm of both prokaryotic and eukaryotic cells and operates without the necessity of oxygen, making it an anaerobic pathway.

Stage I: Preparatory Phase / Energy Investment

• Overall Reaction: Glucose + 2 ATP → 2 Glyc-3P + 2 ADP

• This initial stage encompasses the phosphorylation of glucose and its subsequent conversion into glyceraldehyde 3-phosphate.

  • Importance: Preparing glucose for later energy harvest necessitates an initial investment of ATP. This investment primes the glucose molecule, setting the stage for efficient energy extraction in the subsequent steps.

• Step 1: Glucose to Glucose 6-phosphate

  • Glucose undergoes phosphorylation via ATP, catalyzed by the enzyme hexokinase.

  • ATP → ADP

  • This marks the first priming reaction in the glycolytic pathway.

  • Additional Details: Different tissues express different hexokinase isozymes. For instance, the liver features glucokinase, which exhibits a higher K_m for glucose. This allows the liver to effectively regulate blood glucose levels by modulating glucose metabolism according to the body's needs.

• Step 2: Glucose 6-phosphate to Fructose 6-phosphate

  • Glucose 6-phosphate is isomerized into fructose 6-phosphate by the enzyme phosphohexose isomerase.

  • Significance: This isomerization is crucial as it prepares the molecule for the subsequent phosphorylation step that must occur efficiently for glycolysis to proceed effectively.

• Step 3: Fructose 6-phosphate to Fructose 1,6-bisphosphate

  • Fructose 6-phosphate is phosphorylated using ATP, a reaction catalyzed by phosphofructokinase-1 (PFK-1).

  • ATP → ADP

  • This constitutes the second priming reaction within glycolysis.

  • Regulation: PFK-1 serves as a critical regulatory enzyme, subject to allosteric control by molecules such as ATP, AMP, and fructose 2,6-bisphosphate. These factors modulate enzyme activity based on the energy status and hormonal signals present in the cell.

• Step 4: Cleavage of Fructose 1,6-bisphosphate

  • Fructose 1,6-bisphosphate is cleaved into glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) by the enzyme aldolase.

  • Enzyme Details: Aldolase facilitates the breakdown of a six-carbon sugar into two distinct three-carbon sugars, each of which proceeds through the latter stages of glycolysis.

• Step 5: Isomerization

  • Dihydroxyacetone phosphate is converted into glyceraldehyde 3-phosphate by the enzyme triose phosphate isomerase (TPI).

  • Efficiency: By ensuring that both products from the cleavage of fructose 1,6-bisphosphate are funneled into the same pathway, this step maximizes the efficiency of glycolysis.

Stage II: Payoff Phase

• Overall Reaction: 2 Glyc-3P + 2 NAD+ + 4 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 4 ATP + 2 H2O

• This later stage is defined by substrate-level phosphorylation, a process that directly generates ATP.

  • ATP Production: The payoff phase generates ATP and NADH, effectively capturing the energy initially stored in the glucose molecule. This energy is then available for cellular functions.

High-Energy Phosphate Compounds

• These are compounds with the capacity to undergo substrate-level phosphorylation, directly transferring phosphate groups to ADP to form ATP.

  • Examples: Prominent examples include 1,3-bisphosphoglycerate and phosphoenolpyruvate (PEP), both of which play crucial roles in ATP generation during glycolysis.

Step 1: Glucose to Glucose 6-phosphate

• This reaction is catalyzed by hexokinase.

• During the reaction, ATP is converted to ADP.

• A phosphoryl group is added to glucose, forming glucose 6-phosphate.

  • Importance: This step traps glucose within the cell, as the addition of the phosphate group makes it too polar to cross the cell membrane unaided. Furthermore, it primes glucose, preparing it for subsequent metabolic steps.

Impact of Phosphoryl Group on Membrane Permeability

• The addition of a phosphoryl group significantly reduces the membrane permeability of Glucose 6-phosphate (G6P).

  • Barrier: Due to its negative charge, G6P cannot easily cross the hydrophobic interior of the cell membrane. This effectively commits G6P to further metabolism via glycolysis or other pathways, preventing its exit from the cell.

Hexokinase Mechanism Basics

• Hexokinase relies on magnesium ions to stabilize the triphosphate groups of ATP.

• Shielding the negative charges of the phosphate groups facilitates a nucleophilic attack by a hydroxyl group on glucose.

• This process exemplifies metal-ion catalysis.

• Phosphorylation by hexokinase effectively sequesters hexoses inside the cell.

• Once phosphorylated, glucose is unable to exit the cell through its original transporter.

  • Metal-ion Catalysis: Mg^{2+} plays a pivotal role in facilitating the reaction. It neutralizes the negative charges on ATP, rendering the phosphorus atom more accessible to nucleophilic attack and stabilizing the transition state.

Step 2: Glucose 6-phosphate to Fructose 6-phosphate

• The enzyme phosphohexose isomerase catalyzes this reaction.

• Glucose 6-phosphate is transformed into fructose 6-phosphate via isomerization.

  • Isomerization: Converting an aldose (glucose) to a ketose (fructose) is vital because it sets the stage for phosphorylation at the C-1 position in the subsequent step, which is critical for continuing glycolysis.

Phosphohexose Isomerase Mechanism

• This mechanism entails acid-catalyzed ring opening, followed by base catalysis, and concludes with acid catalysis.

• It also involves the binding of the substrate and the release of the product.

• The reaction proceeds through a cis-Enediolate intermediate.

  • Cis-Enediolate Intermediate: This intermediate is essential for the isomerization process. It facilitates proton transfer and rearrangement of carbon-oxygen bonds, enabling the conversion of glucose 6-phosphate to fructose 6-phosphate.

The Pool of Hexoses

• These intermediates are commonly shared between glycolysis and gluconeogenesis.

• They are interconnected with other pathways, including polysaccharide biosynthesis and the pentose phosphate pathway.

  • Metabolic Crossroads: This highlights the central role of glucose-6-phosphate as a key metabolic intermediate. It can be directed toward various pathways based on the cellular needs and conditions, illustrating the flexibility and interconnectedness of metabolism.

Step 3: Fructose 6-phosphate to Fructose 1,6-bisphosphate

• This reaction is catalyzed by phosphofructokinase-1 (PFK-1).

• ATP is converted to ADP as fructose 6-phosphate is phosphorylated.

• This represents another phosphorylation step in glycolysis.

  • Regulation: PFK-1 is a highly regulated enzyme that serves as a primary control point in glycolysis. It is allosterically modulated by various factors, including AMP, fructose-2,6-bisphosphate (activating), and ATP and citrate (inhibiting). This ensures that glycolysis is responsive to the energy status of the cell and the availability of alternative fuels.

Phosphofructokinase: A Committed Step

• This step is considered a committed step because it directs the pathway irrevocably toward pyruvate production.

• The phosphorylation of glucose commits it to entry into the hexose pool.

• These steps are meticulously regulated to prevent wasteful cycling and ensure appropriate metabolic flux.

• If phosphofructokinase is inhibited, other pathways, such as the pentose phosphate pathway and polysaccharide biosynthesis, become more prominent.

• The energy charge, reflecting the cell's immediate energy requirements, plays a significant role in regulating this step.

• Energy charge serves as an index to gauge the energy status of cells, influencing enzyme activity accordingly.

  • Energy Charge: Elevated ATP levels signify a high energy charge, which inhibits PFK-1, thereby reducing glycolytic flux when energy is abundant. Conversely, low ATP or high AMP levels stimulate PFK-1 to enhance glycolysis and ATP production.

  • Alternative Pathways: When PFK-1 is inhibited, glucose-6-phosphate is diverted to the pentose phosphate pathway. This results in the production of NADPH (used in reductive biosynthesis) and pentose phosphates, essential for nucleotide synthesis.

Step 4: Fructose 1,6-bisphosphate to G3P and DHAP

• This reaction is catalyzed by aldolase.

• Fructose 1,6-bisphosphate is split into glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP).

• Cyclic sugars like fructose can linearize, facilitating this cleavage.

• The cleavage occurs at the 3rd carbon of the ketose sugar, weakening the bond due to polarized effects.

• Although reversible and thermodynamically unfavorable (ΔG° > 0), this reaction proceeds due to a low product-to-reactant ratio maintained by subsequent steps.

  • Mechanism: Aldolase facilitates this cleavage through Schiff base formation with a lysine residue in its active site, stabilizing key intermediates.

  • Thermodynamics: The reaction is driven forward by rapid removal of products, ensuring the continuous conversion of fructose 1,6-bisphosphate into G3P and DHAP.

Aldolase Mechanism

• Fructose-1,6-bisphosphate enters the mechanism in its linear form.

• Approximately 3% of monosaccharides exist in the linear form at any given time.

• The reaction is analogous to non-enzymatic base-catalyzed aldol cleavage but is significantly accelerated by enzymatic catalysis.

• The mechanism involves Schiff base formation, typically involving a lysine residue and a carbonyl group.

• Type II aldolases utilize Zn^{2+} to polarize the carbonyl, aiding in bond cleavage.

  • Schiff Base Formation: This covalent intermediate stabilizes the transition state, reducing the activation energy and facilitating bond cleavage.

Step 5: DHAP to Glyceraldehyde 3-phosphate

• Catalyzed by triose phosphate isomerase (TPI).

• Dihydroxyacetone phosphate (DHAP) is converted to glyceraldehyde 3-phosphate (G3P).

• This step initiates the doubling up, leading to two molecules of glyceraldehyde 3-phosphate for each original glucose molecule.

• Triose phosphate isomerase mechanism involves an enediol intermediate.

  • Efficiency: TPI is considered a kinetically perfect enzyme. Its catalytic efficiency is so high that the reaction rate is limited only by the diffusion of the substrate.

  • Enediol Intermediate: The enediol intermediate stabilizes the transition state, significantly lowering the activation energy and speeding up the interconversion of DHAP and G3P.

Inhibitors

• Potent inhibitors are often designed as transition state analogs, binding with significantly higher affinity than the substrate itself.

  • Medical Applications: Inhibitors of glycolytic enzymes have potential applications as antibacterial or anticancer agents by disrupting energy production in targeted cells.

Step 6: G3P to 1,3-Bisphosphoglycerate

• Catalyzed by glyceraldehyde 3-phosphate dehydrogenase.

• Glyceraldehyde 3-phosphate (G3P) is converted to 1,3-bisphosphoglycerate (1,3-BPG).

• 2NAD^+ is reduced to 2NADH + 2H^+.

• Inorganic phosphate is incorporated into the product.

  • Redox Reaction: This is the first redox reaction in glycolysis, generating NADH, which carries high-energy electrons that can be used to drive ATP synthesis in the electron transport chain.

Glyceraldehyde 3-phosphate Dehydrogenase Mechanism

• The mechanism involves the addition of an active site thiol to the substrate.

• This encompasses substrate binding, thiohemiacetal intermediate formation, dehydrogenation (oxidation), phosphate binding, and product release.

• Forming a high-energy phosphate compound involves coupling the reaction with a high-energy cofactor.

  • Thiohemiacetal Intermediate: This intermediate allows for the oxidation of the aldehyde group of G3P and the subsequent binding of inorganic phosphate, forming a high-energy acyl phosphate bond in 1,3-BPG.

Step 7: 1,3-Bisphosphoglycerate to 3-phosphoglycerate

• Catalyzed by phosphoglycerate kinase.

• 1,3-Bisphosphoglycerate is converted to 3-phosphoglycerate.

• 2ADP is phosphorylated to 2ATP.

• G3P dehydrogenase and phosphoglycerate kinase are coupled reactions.

• G3P dehydrogenase generates a high-energy phosphate compound, while phosphoglycerate kinase removes the phosphoryl group and adds it to ADP, resulting in ATP synthesis (ΔG < 0 overall).

• Coupling is facilitated through direct transfer of 1,3-BPG from one enzyme to another, representing the first instance of substrate-level phosphorylation in glycolysis.

  • Substrate-Level Phosphorylation: This involves the direct transfer of a phosphate group from a substrate molecule (1,3-BPG) to ADP, forming ATP. This is in contrast to oxidative phosphorylation, which occurs in the mitochondria.

  • Coupled Reactions: The energy released from the oxidation of G3P is conserved through the formation of 1,3-BPG, which then directly drives ATP synthesis by phosphoglycerate kinase.

How is 2,3-BPG Made?

• 2,3-BPG serves as an allosteric inhibitor of hemoglobin, stabilizing the T state and promoting oxygen release.

• It forms as a side product of 1,3-BPG.

• Bisphosphoglycerate mutase helps regulate both energy needs within the cell and oxygen delivery throughout the body.

  • Regulation of Hemoglobin: 2,3-BPG binds to hemoglobin, reducing its affinity for oxygen. This facilitates the release of oxygen in tissues where it is most needed, such as during exercise or in hypoxic conditions.

Step 8: 3-phosphoglycerate to 2-phosphoglycerate

• Catalyzed by phosphoglycerate mutase.

• 3-Phosphoglycerate is converted to 2-phosphoglycerate.

• This enzyme is upregulated in some cancers, contributing to the Warburg effect.

  • Warburg Effect: Cancer cells often exhibit increased rates of glycolysis, even in the presence of oxygen (aerobic glycolysis). Upregulation of phosphoglycerate mutase is part of this phenomenon, supporting rapid cell growth and division.

Step 9: 2-phosphoglycerate to phosphoenolpyruvate

• Catalyzed by enolase.

• 2-Phosphoglycerate is converted to phosphoenolpyruvate (PEP).

• 2H_2O is eliminated, forming a high-energy enol phosphate.

• Bacterial enolase is inhibited by fluoride, a mechanism used in some antimicrobial applications.

• PEP represents another high-energy phosphate compound generated during glycolysis.

• PEP completes a pool of interconvertible trioses, similar to the hexose pool in earlier steps.

• Members of this pool are involved in other pathways.

  • e.g., In plants, PEP is involved in the biosynthesis of aromatic amino acids via the Shikimate pathway.

  • Dehydration: Enolase removes a molecule of water from 2-phosphoglycerate, creating a high-energy enol phosphate bond in PEP. This bond is critical for the subsequent ATP synthesis step.

  • Shikimate Pathway: In plants and microorganisms, PEP is a precursor for the synthesis of aromatic amino acids, plant hormones, and various secondary metabolites essential for growth and defense.

Step 10: Phosphoenolpyruvate to Pyruvate

• Catalyzed by pyruvate kinase.

• Phosphoenolpyruvate (PEP) is converted to pyruvate.

• 2ADP is phosphorylated to 2ATP, yielding the second substrate-level phosphorylation event.

• This is an irreversible step that commits the pathway to move away from the triose pool.

• The mechanism involves Mg^{2+} and potassium ions as cofactors.

• Pyruvate kinase is upregulated by fructose-1,6-bisphosphate and PEP and downregulated by ATP and alanine.

• Pyruvate is also utilized in alanine biosynthesis.

  • Irreversible Step: Pyruvate kinase catalyzes the final irreversible step in glycolysis, ensuring unidirectional flow toward pyruvate production under normal cellular conditions.

  • Allosteric Regulation: Pyruvate kinase is allosterically regulated to coordinate its activity with the cell's energy status and metabolic needs. Fructose-1,6-bisphosphate, an earlier glycolytic intermediate, activates the enzyme (feed-forward activation), while ATP and alanine inhibit it (feedback inhibition).

Glycolysis Overview

• Overall reaction: Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O

Regulation of Glycolysis

• Regulation occurs at several key enzymatic steps:

  • Hexokinase - regulated by glucose-6-phosphate.

  • Phosphofructokinase - regulated by ATP, AMP, and fructose 2,6-bisphosphate.

  • Pyruvate Kinase - regulated by ATP, alanine, and fructose 1,6-bisphosphate.

  • Summary: Glycolysis is predominantly controlled at the irreversible steps catalyzed by hexokinase, PFK-1, and pyruvate kinase. These enzymes respond to various signals, thereby maintaining cellular energy homeostasis and coordinating glycolysis with other metabolic pathways.

• These steps are bypassed in gluconeogenesis by:

  • Glucose-6-phosphatase

  • Fructose-1,6-bisphosphatase

  • Pyruvate carboxylase and PEPCK

  • Gluconeogenic Enzymes: These enzymes catalyze bypass reactions that circumvent the irreversible steps of glycolysis, enabling the synthesis of glucose from pyruvate and other precursors, particularly during periods of fasting or starvation.

Why do we need Gluconeogenesis?

• The brain, nervous system, and red blood cells primarily rely on glucose for ATP production.

• Prolonged fasting and intense exercise can deplete glycogen stores, necessitating an alternative glucose source.

• The liver can upregulate gluconeogenesis and export newly synthesized glucose to meet the needs of other tissues.

• Lactate, produced during anaerobic glycolysis, can be converted back to pyruvate and serve as a substrate for gluconeogenesis.

  • Metabolic Context: Gluconeogenesis is an essential metabolic pathway for maintaining blood glucose levels during periods of fasting, starvation, and intense exercise. It ensures that critical organs and tissues have a constant supply of glucose for energy production.

Regulation of Glycolysis and Gluconeogenesis

• Irreversible steps are regulated differently to ensure unidirectional metabolic flux.

• Reversible steps respond to the concentrations of reactants and products.

  • Reciprocal Regulation: Glycolysis and gluconeogenesis are reciprocally regulated to prevent futile cycling and ensure efficient energy utilization. Regulatory mechanisms coordinate these pathways, activating one while inhibiting the other, depending on the cellular energy status and hormonal signals.

Regulation of Hexokinase

• Mammals express four distinct isozymes of hexokinase (I, II, III, and IV).

• Type II predominates in muscle tissue and is subject to inhibition by glucose-6-phosphate.

Type II vs Type IV Hexokinase

• Type IV hexokinase, also known as glucokinase, is primarily found in the liver.

• Glucokinase is not inhibited by glucose-6-phosphate but is induced by insulin.

• Key characteristics:

  • Hexokinase (II) displays non-specificity toward hexoses.

  • The K_m is approximately 100 \,
    μM.

  • It is inhibited by G-6-P.

  • Glucokinase (IV) demonstrates specificity solely for glucose.

  • The K_m is approximately 5 \, mM.

  • It is not inhibited by G-6-P.

  • Km Values: The differences in K_m values between hexokinase II and glucokinase reflect their distinct roles. Glucokinase's lower affinity ensures that the liver only phosphorylates glucose when it is abundant, preventing excessive glucose consumption by the liver.

Rationale

• Hexokinase (II) is present in all tissues:

  • Rationale: This isozyme functions to metabolize blood glucose for energy production and to trap glucose within the cell. Its activity is inhibited when cellular energy levels are high and glucose metabolism is not immediately required.

• Glucokinase (IV) is exclusive to the liver:

  • Rationale: This isozyme plays a crucial role in maintaining moderate blood glucose levels and sequestering excess glucose for glycogen production. Mutations that impair glucokinase function can adversely affect blood glucose homeostasis, leading to hyperglycemia or other metabolic disturbances.

• These distinct hexokinase isozymes reflect the specialized roles of the respective organs and tissues:

  • Muscles consume glucose primarily for energy production to support physical activity.

  • The liver is responsible for maintaining glucose homeostasis, ensuring a stable supply of glucose to other tissues.

Glucose Levels → Outcomes

• High glucose levels trigger the following responses:

  • Induction of glucokinase production and elevated activity in the liver, resulting in a reduction of blood glucose levels.

  • Increased glucose transport into body tissues, further lowering blood glucose concentrations.

  • Enhanced glycolysis in the liver, promoting glucose utilization and reducing its concentration.

• Low glucose levels elicit these counter-regulatory mechanisms:

  • Glycogen breakdown by the liver, releasing glucose into the bloodstream and raising blood glucose levels.

  • Increased gluconeogenesis in the liver, synthesizing new glucose and elevating blood glucose levels.

  • Decreased glycolysis in the liver, conserving glucose and prioritizing its release into the circulation.

• These intricate processes are orchestrated by hormones such as insulin and glucagon.

  • Hormonal Control: Insulin and glucagon serve as key hormonal regulators, coordinating glucose metabolism by modulating the expression and activity of pivotal enzymes involved in glycolysis and gluconeogenesis. This ensures a balanced response to varying physiological conditions, such as feeding and fasting.

Glucose Homeostasis (Insulin/Glucagon)

• Low glucose levels (↑glucagon): Increased gluconeogenesis → release of blood glucose

• High glucose levels (↑insulin): Stimulation of glycogen formation involves upregulation of glucokinase and upregulation of glucose transporters in tissue cells

  • Hormonal Regulation: Insulin facilitates glucose uptake and utilization by promoting the translocation of glucose transporters to the cell surface and stimulating glycogen synthesis. Conversely, glucagon stimulates glucose production and release by activating glycogen breakdown and gluconeogenesis in the liver.

Regulation of Phosphofructokinase

• PFK-1 is the primary control point within the glycolytic pathway, exerting significant influence over metabolic flux.

• The enzyme is activated by the need for ATP, reflecting the cellular energy charge and driving glycolysis when energy is scarce.

• PFK-1 is also activated by the specific demand for glycolysis, indicated by elevated citrate levels that signal a need to process more glucose.

• Fructose 2,6-bisphosphate serves as a potent allosteric activator of PFK-1, generated from fructose 6-phosphate in a feed-forward mechanism.

  • Fructose 2,6-Bisphosphate: This activator markedly enhances PFK-1 activity, thereby increasing glycolytic flux in response to hormonal cues and shifting the balance away from gluconeogenesis.

Fructose 2,6-bisphosphate Regulation

• Fructose 2,6-bisphosphate also inhibits gluconeogenesis through the inhibition of fructose 1,6-bisphosphatase.

• High levels of F26BP promote glycolysis while simultaneously inhibiting gluconeogenesis.

• Conversely, low levels of F26BP reduce the rate of glycolysis and facilitate gluconeogenesis.

• The levels of F26BP correspond directly to the availability of glucose, providing a sensitive mechanism for metabolic adjustment.

Fructose 2,6-bisphosphate Production

• Fructose-6-phosphate is converted to fructose-2,6-bisphosphate through the action of PFK-2, particularly in the liver.

• F26BP can be reverted to F6P by FBPase-2, which is the phosphorylated form of PFK-2.

• The equilibrium between F6P and F26BP is governed by the phosphorylation state of PFK-2.

  • Bifunctional Enzyme: PFK-2/FBPase-2 functions as a bifunctional enzyme, uniquely regulating the levels of fructose 2,6-bisphosphate. This, in turn, creates a direct link between glycolysis and gluconeogenesis through a single regulatory molecule responsive to hormonal and metabolic signals.

Regulation of Phosphofructokinase-2

• The kinases and phosphatases governing PFK-2 phosphorylation are themselves under the control of the insulin/glucagon signaling system.

• Insulin promotes the dephosphorylation of PFK-2, leading to increased F26BP production, upregulation of glycolysis, and inhibition of gluconeogenesis.

• Glucagon promotes the phosphorylation of PFK-2, resulting in decreased F26BP levels, downregulation of glycolysis, and a relative increase in gluconeogenesis.

  • Hormonal Control: Insulin activates PFK-2, thereby elevating F2,6-BP levels. This action stimulates glycolysis and reduces gluconeogenesis. Conversely, glucagon activates FBPase-2, lowering F2,6-BP concentrations and inhibiting glycolysis while promoting gluconeogenesis.

Regulation of Pyruvate Kinase

• The regulatory mechanisms governing pyruvate kinase align with those of the previously discussed enzymes.

• The cellular energy charge exerts a substantial influence, serving as an allosteric inhibitor when ATP levels are high.

• The insulin/glucagon system participates in the activation/deactivation of pyruvate kinase through dephosphorylation/phosphorylation mechanisms.

• Liver isozymes and general isozymes of pyruvate kinase exhibit differing behaviors in response to regulatory signals.

• Insulin promotes the activity of pyruvate kinase by triggering its dephosphorylation, leading to increased enzymatic activity and glycolytic flux. This ensures efficient glucose utilization when glucose levels are high.

  • Isozymes: Tissue-specific isozymes of pyruvate kinase in the liver (L-form) and other tissues (M-form) exhibit differing regulatory properties.

    • The L-form is subject to more complex regulation, including allosteric control and covalent modification (phosphorylation/dephosphorylation), enabling the liver to fine-tune glycolytic activity in response to hormonal signals and metabolic conditions.

  • Allosteric Regulation: Liver pyruvate kinase is allosterically activated by fructose-1,6-bisphosphate (F-1,6-BP), an intermediate of glycolysis.

    • This feed-forward activation ensures that increased glycolytic flux leads to enhanced pyruvate kinase activity, further stimulating the pathway.

    • Conversely, it is inhibited by ATP and alanine, providing feedback control based on the energy status and amino acid levels in the cell.

  • Hormonal Regulation: The L-form of pyruvate kinase is regulated by phosphorylation and dephosphorylation in response to insulin and glucagon signaling.

    • Insulin promotes dephosphorylation, activating the enzyme and increasing glycolytic flux.

    • Glucagon promotes phosphorylation via cAMP-dependent protein kinase A (PKA), inactivating the enzyme and reducing glycolytic flux.

    • This hormonal regulation allows the liver to adjust glucose metabolism according to blood glucose levels.

  • M-Form Regulation: The M-form of pyruvate kinase, primarily found in muscle and other tissues, is less sensitive to allosteric and hormonal regulation compared to the L-form.

    • This ensures that glycolysis proceeds at a relatively constant rate in these tissues, independent of hormonal fluctuations. However, it is still subject to feedback inhibition by ATP.

  • Significance: The differential regulation of pyruvate kinase isozymes in the liver and other tissues reflects the specialized roles of these organs in glucose metabolism.