Glycolysis, Gluconeogenesis, & Glycogen Metabolism Notes

Glycolysis, Gluconeogenesis, & Glycogen Metabolism

Student Learning Goals for Glucose Metabolism

• To describe the substrates, products, and chemical reactions for each step of glycolysis.

• To differentiate fermentation under anaerobic conditions.

• To describe the synthesis of glucose from simpler compounds: gluconeogenesis.

• To describe the substrates, products, and reactions of gluconeogenesis.

• To identify enzymes that are unique to glycolysis.

• To identify and recall enzymes unique to gluconeogenesis.

• To define and point towards allosteric regulatory steps that decide the fate of glycolysis versus gluconeogenesis.

Key Concepts of Glycolysis

• Metabolic Intermediates: The glycolysis pathway provides metabolic intermediates essential for other biosynthetic processes.

• Location: It occurs exclusively in the cytoplasm of the cell.

• Universality and Ancient Origin: Glycolysis is a universal and ancient mechanism for harvesting energy from glucose, fundamental for generating quick cellular energy.

• Oxygen Independence: It is particularly vital in tissues or organisms lacking oxygen, as it can generate ATP rapidly without molecular oxygen.

• Essential Role: Due to its ability to produce ATP without oxygen, glycolysis is essential for various organisms and cell types, from red blood cells to actively contracting muscles.

Four Major Pathways of Glucose Utilization

Glucose can be utilized through four main pathways:

1. Storage: Converted into glycogen, starch, or sucrose.

2. Synthesis: Used for the synthesis of structural polymers, such as extracellular matrix components and cell wall polysaccharides.

3. Oxidation via Pentose Phosphate Pathway: Produces ribose 5-phosphate, crucial for nucleotide synthesis, and NADPH.

4. Oxidation via Glycolysis: Breaks down glucose into pyruvate, generating ATP and NADH.

Glycolysis: Location and Oxygen Requirement

• Cellular Location: Glycolysis takes place in the cytosol of the cell.

• Oxygen Requirement: Glycolysis itself does not require oxygen. It is the first step of cellular respiration and proceeds under both aerobic and anaerobic conditions.

• Fate of Pyruvate in Anaerobic Conditions (Fermentation):

  • In animals (e.g., actively contracting muscles, red blood cells), pyruvate is converted to lactate. This process regenerates $NAD^+$ from NADH, sustaining glycolysis.

  • In yeast, pyruvate is converted to ethanol and $CO_2$. This also regenerates $NAD^+$.

• Fate of Pyruvate in Aerobic Conditions:

  • Pyruvate enters the mitochondria for further aerobic metabolism, being converted to acetyl co-A, which then enters the Krebs Cycle and the electron transport chain, leading to significant additional ATP production.

• Net ATP Production: Under either aerobic or anaerobic conditions, glycolysis itself yields a net of 2 ATP per glucose molecule.

General Overview of Glycolysis

• Sequence of Reactions: Glycolysis involves a sequence of 10 enzyme-catalyzed reactions.

• Location: These reactions occur in the cytoplasm of almost all living cells.

• First Step of Cellular Respiration: It is the initial stage of cellular respiration, operating under both aerobic and anaerobic environments.

Glycolysis: Glucose Molecule Split

• Glycolysis literally means "sugar splitting." Enzymes within the cytosol break apart a 6-carbon glucose molecule into two 3-carbon molecules of pyruvate.

• The energy released during the breaking of chemical bonds in glucose is transferred to carrier molecules: ATP (adenosine triphosphate) and NADH (nicotinamide adenine dinucleotide).

• NADH temporarily holds small amounts of energy that can be subsequently used to synthesize more ATP.

Net Gain Following Glycolysis

• Per glucose molecule processed, the net gain is:

  • $2$ ATP molecules

  • $2$ NADH molecules

  • $2$ pyruvate molecules

• Key Phases:

  • Initial Phase (Energy Investment): This phase requires an input of $2$ ATP molecules to phosphorylate glucose, preparing it for breakdown.

  • Later Steps (Energy Payoff): This phase generates $4$ ATP molecules (a net gain of $2$ ATP after the investment), $2$ NADH molecules, and cleaves the six-carbon glucose into $2$ three-carbon pyruvates.

Glucose as an Important Fuel

• Brain Fuel: In mammals, glucose is the sole fuel that the brain utilizes under non-starvation conditions.

• Red Blood Cell Fuel: Glucose is the only fuel that red blood cells can use under any circumstances.

Why Red Blood Cells (RBCs) Use Only Glucose for Energy

• Lack of Organelles: Mature red blood cells are anucleate (lack a nucleus) and lack most other organelles, critically including mitochondria.

• Reliance on Anaerobic Glycolysis: Therefore, they rely entirely on anaerobic glycolysis for their energy production. This adaptation allows RBCs to efficiently transport oxygen throughout the body without consuming it for their own energy needs.

Glycolysis vs. Gluconeogenesis

• Gluconeogenesis Defined: This is the metabolic process by which glucose is synthesized from non-carbohydrate precursors, such as pyruvate, lactate, and certain amino acids.

• Not Simple Reversals: Glycolysis and gluconeogenesis are not simply the reverse of each other. While many steps are reversible and use the same enzymes, there are several irreversible steps that require distinct enzymes in each pathway.

• Reciprocal Regulation: These two pathways are reciprocally regulated, meaning that when one pathway is highly active, the other is relatively inactive, preventing a 'futile cycle' where energy would be wasted.

Enzymes in Glycolysis

Glycolysis involves several classes of enzymes:

• Kinase: Catalyzes the transfer of a phosphoryl group, typically from ATP.

• Mutase: Catalyzes the shift of a phosphoryl group from one atom to another within the same molecule.

• Isomerase: Catalyzes the conversion of one isomer to another (e.g., aldose to ketose).

• Aldolase: Catalyzes the cleavage of a C-C bond (aldol cleavage).

• Dehydrogenase: Catalyzes oxidation-reduction reactions, often involving the transfer of electrons to $NAD^+$ or $NADP^+$.

Metabolic Goals of Glycolysis

1. ATP Generation: To extract the free energy released during the catabolism of glucose and store it directly in the form of ATP.

2. NADH Production: To extract and store electrons, primarily as NADH. This NADH can then be used either to generate more ATP through oxidative phosphorylation or to provide reducing power for biosynthesis in the form of NADPH (after conversion).

3. Intermediate Provision: To provide metabolic intermediates that serve as precursors for other biosynthetic pathways.

4. Pyruvate Production: To produce pyruvate, which is a key molecule for the next stages of energy production (e.g., the citric acid cycle under aerobic conditions).

Detailed Steps of Glycolysis

Glycolysis consists of 10 enzyme-catalyzed reactions:

• Step 1: Hexokinase Reaction

  • Reaction: Glucose $<br>ightarrow$ Glucose 6-phosphate

  • Enzyme: Hexokinase (or glucokinase in the liver)

  • Characteristics: This step is irreversible. It phosphorylates glucose at the expense of ATP, trapping it inside the cell and initiating glycolysis.

• Step 2: Phosphoglucose Isomerase Reaction

  • Reaction: Glucose 6-phosphate $<br>ightarrow$ Fructose 6-phosphate

  • Enzyme: Phosphoglucose isomerase

  • Characteristics: This is a freely reversible isomerization, converting an aldose (glucose 6-phosphate) into a ketose (fructose 6-phosphate).

• Step 3: Phosphofructokinase (PFK) Reaction

  • Reaction: Fructose 6-phosphate $<br>ightarrow$ Fructose 1,6-bisphosphate

  • Enzyme: Phosphofructokinase-1 (PFK-1)

  • Characteristics: This is an irreversible and highly regulated reaction, often referred to as the "committing step" of glycolysis. It involves the addition of a second phosphate group, further trapping the carbohydrate within the fructose form in the cell. PFK-1 is an allosteric enzyme.

• Step 4: Aldolase Reaction

  • Reaction: Fructose 1,6-bisphosphate $<br>ightarrow$ Glyceraldehyde 3-phosphate (GAP) + Dihydroxyacetone phosphate (DHAP)

  • Enzyme: Aldolase

  • Characteristics: This step is reversible and involves the cleavage of fructose 1,6-bisphosphate into two three-carbon molecules.

• Step 5: Triose Phosphate Isomerase (TPI) Reaction

  • Reaction: Dihydroxyacetone phosphate (DHAP) $<br>ightarrow$ Glyceraldehyde 3-phosphate (GAP)

  • Enzyme: Triose phosphate isomerase (TPI)

  • Characteristics: This step is reversible. Only GAP can be directly processed to pyruvate. TPI interconverts DHAP into GAP, ensuring that all 6 carbons of the original glucose molecule can proceed through the rest of glycolysis. Deficiency of TPI is lethal.

• Step 6: Glyceraldehyde 3-phosphate Dehydrogenase Reaction

  • Reaction: Glyceraldehyde 3-phosphate $<br>ightarrow$ 1,3-Bisphosphoglycerate

  • Enzyme: Glyceraldehyde 3-phosphate dehydrogenase

  • Characteristics: This step is reversible. GAP is oxidized and phosphorylated to 1,3-bisphosphoglycerate, a high phosphoryl-transfer potential compound. This reaction also produces NADH from $NAD^+$.

• Step 7: Phosphoglycerate Kinase Reaction

  • Reaction: 1,3-Bisphosphoglycerate $<br>ightarrow$ 3-Phosphoglycerate

  • Enzyme: Phosphoglycerate kinase

  • Characteristics: This step is reversible. 1,3-bisphosphoglycerate, an energy-rich molecule, transfers its phosphoryl group to ADP, generating ATP in a process known as substrate-level phosphorylation.

• Step 8: Phosphoglycerate Mutase Reaction

  • Reaction: 3-Phosphoglycerate $<br>ightarrow$ 2-Phosphoglycerate

  • Enzyme: Phosphoglycerate mutase

  • Characteristics: This step is freely reversible and involves the intramolecular shift of the phosphate group.

• Step 9: Enolase Reaction

  • Reaction: 2-Phosphoglycerate $<br>ightarrow$ Phosphoenolpyruvate (PEP)

  • Enzyme: Enolase

  • Characteristics: This step is reversible. It involves the removal of a water molecule, creating an enol phosphate, Phosphoenolpyruvate (PEP), which has a very high phosphoryl-transfer potential.

• Step 10: Pyruvate Kinase Reaction

  • Reaction: Phosphoenolpyruvate (PEP) $<br>ightarrow$ Pyruvate

  • Enzyme: Pyruvate kinase

  • Characteristics: This step is irreversible. PEP transfers its high-energy phosphate group to ADP, generating ATP (another substrate-level phosphorylation). The large driving force for this reaction comes from the tautomerization of the enol form of pyruvate to the more stable keto form.

High Phosphoryl-Transfer Potential of Phosphoenolpyruvate (PEP)

• The exceptionally high phosphoryl-transfer potential of PEP is primarily attributed to the large negative free energy change associated with the subsequent enol-ketone conversion of pyruvate.

What Happens to Pyruvate?

After glycolysis, the fate of pyruvate depends on the availability of oxygen:

• Regeneration of $NAD^+$: The conversion of glucose to pyruvate generates ATP, but for continued ATP synthesis through glycolysis, NADH must be reoxidized to $NAD^+$. This vital coenzyme is derived from vitamin niacin (B3).

• Anaerobic Conditions (Fermentation):

  • If oxygen is unavailable, $NAD^+$ is regenerated by the conversion of pyruvate to lactic acid (in animals, via lactate dehydrogenase) or to ethanol and carbon dioxide (in yeast, via pyruvate decarboxylase and alcohol dehydrogenase). Louis Pasteur termed this process fermentation, or "life without air."

  • This regeneration of $NAD^+$ is crucial for maintaining redox balance and sustaining glycolysis under anaerobic conditions.

• Aerobic Conditions:

  • If oxygen is present, pyruvate enters the mitochondria and undergoes further oxidation.

  • It is converted to acetyl-CoA by the pyruvate dehydrogenase complex.

  • Acetyl-CoA then enters the citric acid cycle, leading to the production of far more energy, including many additional molecules of ATP via oxidative phosphorylation.

Glycolysis vs. Gluconeogenesis: Irreversible Steps

Glycolysis and gluconeogenesis are reciprocally regulated. The irreversible steps of one pathway are bypassed by different enzymes and reactions in the other.

Irreversible Steps of Glycolysis

These steps have highly negative free energy changes (box[5px,border:2px solid black]{oldsymbol{ ext{ΔG}}}) and are far from equilibrium, making them effectively one-way. They are coupled with ATP hydrolysis or formation.

• Step 1: Glucose $ightarrow$ Glucose-6-phosphate

  • Enzyme: Hexokinase (or glucokinase in liver)

• Step 3: Fructose-6-phosphate $ightarrow$ Fructose-1,6-bisphosphate

  • Enzyme: Phosphofructokinase-1 (PFK-1)

• Step 10: Phosphoenolpyruvate (PEP) $ightarrow$ Pyruvate

  • Enzyme: Pyruvate kinase

Irreversible Steps of Gluconeogenesis (Bypassing Glycolysis Steps)

These steps are catalyzed by different enzymes to overcome the energetic barriers of the irreversible glycolytic reactions, preventing a futile cycle and allowing strict regulation of glucose production and breakdown.

• Bypass of Pyruvate Kinase (Glycolysis Step 10): Pyruvate $ightarrow$ Phosphoenolpyruvate (PEP)

  • This occurs in two steps, consuming ATP and GTP:

    1. Pyruvate $ightarrow$ Oxaloacetate: Catalyzed by Pyruvate carboxylase. This reaction requires ATP and $CO_2$ and occurs in the mitochondria.

    2. Oxaloacetate $ightarrow$ PEP: Catalyzed by PEP carboxykinase (PCK). This reaction requires GTP and releases $CO_2$ (in some species, can occur in mitochondria or cytosol).

• Bypass of Phosphofructokinase-1 (Glycolysis Step 3): Fructose-1,6-bisphosphate $ightarrow$ Fructose-6-phosphate

  • Enzyme: Fructose-1,6-bisphosphatase (FBPase-1). This is a hydrolysis reaction that removes a phosphate group, releasing inorganic phosphate ($P_i$).

• Bypass of Hexokinase (Glycolysis Step 1): Glucose-6-phosphate $ightarrow$ Glucose

  • Enzyme: Glucose-6-phosphatase (G6PC). This is also a hydrolysis reaction, removing the phosphate group to release free glucose.

  • Location: This enzyme is predominantly found in the liver and, to a lesser extent, the kidney. Its presence in these organs allows them to release newly synthesized glucose into the bloodstream, a metabolic duty crucial for maintaining blood glucose homeostasis. In other cells, glucose-6-phosphate cannot leave the cell.

Regulation of Glycolysis and Gluconeogenesis

• Pathway Reciprocity: Within a cell, glycolysis and gluconeogenesis are reciprocally regulated; when one pathway is active, the other is inhibited.

• Enzyme Control: The amounts and activities of the distinctive enzymes in each pathway are tightly controlled to ensure that both are not highly active simultaneously.

• Substrate/Product Concentration: The rate of glycolysis is influenced by glucose concentration, while gluconeogenesis is affected by the concentration of precursors like lactate.

• Energy Status (Reciprocal Regulation): This is a major factor: when the cell requires energy (low ATP, high AMP), glycolysis predominates. Conversely, when there is a surplus of energy (high ATP), gluconeogenesis takes over to store glucose or convert precursors into glucose.

Regulation of Phosphofructokinase-1 (PFK-1)

• Rate-Limiting Enzyme: PFK-1 is the main rate-limiting enzyme of glycolysis. It regulates the step that commits glucose to breakdown.

• Allosteric Regulation: PFK-1 is highly regulated allosterically, primarily by the cell's energy status:

  • Activation: Activated when energy is low (high AMP concentration).

  • Inhibition: Inhibited when energy is high (high ATP concentration) and by citrate.

• Fructose 2,6-bisphosphate (F-2,6-BP):

  • Potent Allosteric Activator: F-2,6-BP is a powerful allosteric activator of PFK-1.

  • Mechanism: It increases PFK-1’s affinity for its substrate (fructose 6-phosphate) and reduces the enzyme's inhibition by ATP.

  • Hormonal Control: Its levels are hormonally controlled:

    • Insulin: High insulin levels stimulate the production of F-2,6-BP, promoting glycolysis.

    • Glucagon: High glucagon levels decrease F-2,6-BP, inhibiting glycolysis and favoring gluconeogenesis.

  • Dedicated Synthesis: F-2,6-BP is formed by a separate bifunctional enzyme, PFK-2/FBPase-2, outside the main glycolytic pathway. This allows precise control over its levels, enabling the cell to rapidly switch between glycolysis and gluconeogenesis based on energy needs and hormonal signals.

  • Metabolic Signal: F-2,6-BP acts as a crucial metabolic signal, indicating abundant glucose and energy availability, thus activating glucose breakdown.

Glycogen Metabolism

Glycogen Metabolism Learning Goals

• To describe the substrates, products, and reactions of glycogen synthesis and degradation.

• To compare the processes of glycogen synthesis (glycogenesis) and glycogen degradation (glycogenolysis).

• To describe the substrates, products, and reactions of the pentose phosphate pathway.

Energy Storage in Mammals

Energy in mammals is stored in three primary forms:

• Serum Glucose: Approximately $40$ kcal

• Glycogen: About $600$ kcal (stored mainly in muscle and liver)

• Fat: Approximately $100,000$ kcal (the largest reserve)

Glycogen Structure

• Polymer: Glycogen is a highly branched homopolymer composed entirely of glucose units.

• Location: It is present in the cytoplasm of all tissues, with the largest stores found in the liver and skeletal muscle.

• Linkages: Glucose units are linked by:

  • oldsymbol{ ext{ extalpha}}-1,4 glycosidic bonds: Forming the linear chains.

  • oldsymbol{ ext{ extalpha}}-1,6 glycosidic bonds: Forming branches (crosslinks, occurring approximately every 8-12 glucose residues).

• Non-reducing Ends: These are the ends of glycogen molecules where the hydroxyl (OH group) on the anomeric carbon (C1) is not involved in a glycosidic linkage with another glucose molecule. Glycogen has many non-reducing ends.

Significance of Glycogen Branching

• Increased Accessibility: The extensive branching significantly increases the number of non-reducing ends available on the glycogen molecule.

• Simultaneous Addition/Removal: This allows for rapid and simultaneous addition (during synthesis) or removal (during degradation) of many glucose units, making glycogen a highly efficient and quickly mobilizable energy store.

Glycogen Roles

• Liver Glycogen: The liver breaks down its stored glycogen (a process called glycogenolysis) and releases free glucose into the bloodstream. This glucose then provides energy for other cells and tissues throughout the body, especially the brain.

• Muscle Glycogen: Muscle glycogen stores are primarily mobilized to provide immediate energy for muscle contraction, serving the muscle's own energy demands.

Glycogen Breakdown (Glycogenolysis)

• Key Enzyme: Glycogen phosphorylase is the rate-limiting enzyme in glycogen breakdown.

• Reaction: It cleaves glucose units from the non-reducing ends of glycogen by the addition of inorganic phosphate ($P_i$), a process called phosphorolysis, to yield glucose 1-phosphate.

  • Glycogen ($n$ residues) + $P_i <br>ightarrow$ Glucose 1-phosphate + Glycogen ($n-1$ residues)

• Subsequent Steps:

  1. Glucose 1-phosphate is converted to glucose 6-phosphate by phosphoglucomutase.

  2. In the liver (and kidney), glucose 6-phosphate is then dephosphorylated to free glucose by glucose-6-phosphatase. This free glucose can then exit the cell and enter the bloodstream.

Glycogen Synthesis (Glycogenesis)

• Precursor: Glycogen is a polymer formed from glucose-1-phosphate monomers.

• Activated Glucose: Glucose 1-phosphate is first activated by reacting with UTP (uridine triphosphate) to form UDP-glucose, releasing pyrophosphate ($PP_i$).

• Key Enzyme: Glycogen synthase adds glucose units from UDP-glucose to the non-reducing ends of a growing glycogen polymer.

  • UDP-Glucose + Glycogen ($n$ residues) $<br>ightarrow$ UDP + Glycogen ($n+1$ residues)

Regulation of Glycogen Metabolism

• Reciprocal Control: The synthesis and degradation of glycogen are tightly regulated in a reciprocal manner to prevent futile cycling.

• Enzyme Phosphorylation: Kinases add phosphate groups to enzymes, altering their activity, while phosphatases remove them.

  • Glycogen Phosphorylase: Generally active when phosphorylated, promoting glycogen breakdown (e.g., in response to low blood glucose or energy need).

  • Glycogen Synthase: Generally active when dephosphorylated, promoting glycogen synthesis (e.g., in response to adequate blood glucose and no immediate energy need).

• Blood Glucose Regulation: Blood glucose levels primarily regulate liver glycogen metabolism to maintain systemic glucose homeostasis.

The Pentose Phosphate Pathway (PPP)

Fates of Glucose 6-Phosphate

After the degradation of stored glycogen, the resulting glucose 6-phosphate has multiple fates:

• It can enter glycolysis for cellular respiration to produce immediate energy.

• It can enter an alternative pathway known as the Pentose Phosphate Pathway (PPP).

Overview of the Pentose Phosphate Pathway

• Parallel Pathway: The PPP is a metabolic pathway that runs parallel to glycolysis, primarily involved in glucose breakdown.

• Main Products:

  1. NADPH: A crucial helper molecule for anabolic reactions (e.g., fatty acid synthesis) and for protecting cells from oxidative damage by reducing reactive oxygen species.

  2. Ribose 5-phosphate: A 5-carbon sugar essential for the synthesis of DNA, RNA, ATP, NADH, FADH2, and coenzyme A.

• Two Main Phases:

  1. Oxidative Phase: This phase produces NADPH and ribulose 5-phosphate ($CO_2$ is also released).

  2. Non-oxidative Phase: This phase interconverts various sugars, including ribose 5-phosphate, and can convert excess pentose phosphates back into glycolytic intermediates (fructose 6-phosphate and glyceraldehyde 3-phosphate) that the cell can use for energy.

• Importance: The PPP is particularly important in cells that require significant NADPH (e.g., red blood cells for antioxidant defense, liver for fatty acid synthesis) and cells undergoing rapid division that need to synthesize new nucleotides (e.g., bone marrow, cancerous cells).

Oxidative Reactions of the Pentose Phosphate Pathway (NADPH Production)

• These reactions are metabolically irreversible and involve the transfer of hydride ions to $NADP^+$.

  1. Glucose 6-phosphate $ightarrow$ 6-Phosphoglucono-oldsymbol ext{ extdelta}-lactone

     • Enzyme: Glucose-6-phosphate dehydrogenase

     • Product: Produces the first molecule of NADPH.

  2. 6-Phosphoglucono-oldsymbol ext{ extdelta}-lactone $ightarrow$ 6-Phosphogluconate

     • Enzyme: 6-phosphogluconolactonase

     • Characteristics: Hydrolysis reaction.

  3. 6-Phosphogluconate $ightarrow$ Ribulose 5-phosphate

     • Enzyme: 6-phosphogluconate dehydrogenase

     • Products: Produces the second molecule of NADPH and releases $CO_2$.

Ribose 5-Phosphate

• Precursor: Ribose 5-phosphate, formed from ribulose 5-phosphate, is a direct precursor of the ribose unit found in nucleotides (DNA, RNA).

• Pathway End: Its production commonly marks the functional end of the oxidative phase of the pentose phosphate pathway.

• Ribose vs. Deoxyribose: Ribonucleotide reductase converts ribose to deoxyribose by removing an oxygen atom. Ribose is the sugar in RNA, while deoxyribose (meaning "missing an oxygen") is the sugar in DNA.