# Lecture 5: Carbohydrate Metabolism I - Glycolysis ## Unit 1.1: Foundations of Medicine & Medical Sciences --- ## Learning Objectives By the end of this lecture, students will be able to: 1. Outline the complete glycolysis pathway, identifying all substrates, products, and enzymes for each step 2. Identify the three irreversible regulatory enzymes of glycolysis and their allosteric regulators 3. Explain the net ATP yield from glycolysis under aerobic and anaerobic conditions 4. Describe the fate of pyruvate under aerobic and anaerobic conditions 5. Explain the role of glycolysis in different tissues and clinical conditions 6. Identify inherited enzyme deficiencies affecting glycolysis and their clinical presentations --- ## Lecture Content ### I. Overview of Glycolysis Glycolysis—literally "glucose splitting"—stands as the most ancient and universal metabolic pathway. Present in virtually all living cells, from bacteria to human neurons, glycolysis extracts energy from glucose through a sequence of ten enzyme-catalyzed reactions. The pathway occurs entirely in the cytoplasm and requires no oxygen, making it the sole source of ATP for cells lacking mitochondria and the emergency energy source when oxygen becomes limiting. The overall reaction captures the essence of the pathway: one molecule of glucose (a six-carbon sugar) is converted to two molecules of pyruvate (a three-carbon compound), with the net production of two ATP molecules and two NADH molecules. The chemical equation summarizes this transformation: Glucose + 2 NAD⁺ + 2 ADP + 2 Pᵢ → 2 Pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 H₂O. #### Phases of Glycolysis The pathway divides naturally into two phases with distinct energetic profiles. The investment phase (steps 1-5) consumes ATP to activate and rearrange glucose. Two ATP molecules are spent phosphorylating the sugar, and the six-carbon fructose-1,6-bisphosphate is cleaved into two three-carbon molecules: dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. These interconvertible trioses represent the branching point where one glucose molecule becomes two identical metabolic units. The payoff phase (steps 6-10) harvests the energy stored in these phosphorylated three-carbon molecules. Each glyceraldehyde-3-phosphate molecule generates one NADH and two ATP molecules through substrate-level phosphorylation. Since two triose phosphates are processed per glucose, the payoff phase produces four ATP and two NADH molecules—yielding a net gain of two ATP and two NADH after subtracting the initial investment. Panel A: Investment phase showing glucose hexagon with steps 1 and 3 highlighted by red arrows indicating ATP consumption. Panel B: Step 4 cleavage producing two 3-carbon triangular structures with division line separating phases. Panel C: Payoff phase with parallel pathways (x2 notation) from glyceraldehyde-3-phosphate showing green arrows at steps 6, 7, 10 for NADH and ATP production. Panel D: Net energy balance summary box showing -2 ATP invested, +4 ATP produced, net +2 ATP + 2 NADH per glucose. --- ### II. Investment Phase (Steps 1-5) The investment phase prepares glucose for energy extraction by adding phosphate groups that trap the sugar within the cell and destabilize its bonds, making subsequent cleavage possible. #### Step 1: Hexokinase/Glucokinase Reaction The first step irreversibly phosphorylates glucose to glucose-6-phosphate, consuming one ATP molecule. This reaction accomplishes two essential tasks: it traps glucose within the cell (phosphorylated sugars cannot cross the plasma membrane) and commits glucose to further metabolism. The large negative free energy change (ΔG = −16.7 kJ/mol) makes this step effectively irreversible under physiological conditions. Two different enzymes catalyze this reaction in different tissues, reflecting their distinct metabolic roles. Hexokinase operates in most tissues with a very low Km (approximately 0.1 mM), meaning it efficiently captures glucose even at low concentrations and becomes saturated at normal blood glucose levels. This enzyme is inhibited by its product, glucose-6-phosphate, providing feedback regulation that prevents excessive glucose phosphorylation when downstream pathways are backed up. Glucokinase (also called hexokinase IV) functions primarily in liver and pancreatic beta cells. Its high Km (approximately 10 mM) means it operates efficiently only when glucose concentrations rise above normal blood levels. Glucokinase is not inhibited by glucose-6-phosphate, allowing the liver to continue extracting glucose from portal blood after meals. In pancreatic beta cells, glucokinase serves as a glucose sensor, linking blood glucose concentration to insulin secretion. #### Step 2: Phosphoglucose Isomerase This reversible reaction converts the aldose glucose-6-phosphate to the ketose fructose-6-phosphate. The isomerization prepares the molecule for phosphorylation at carbon 1, which will create a symmetrical molecule that can be cleaved into two identical three-carbon fragments. #### Step 3: Phosphofructokinase-1 (PFK-1) — The Rate-Limiting Step The phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate represents the committed step of glycolysis—the point of no return. This reaction, catalyzed by phosphofructokinase-1 (PFK-1), is irreversible and serves as the most important regulatory point in the pathway. By controlling PFK-1 activity, cells can rapidly adjust glycolytic flux in response to energy demands. PFK-1 is an allosteric enzyme exquisitely sensitive to the cell's energy status. When energy is depleted (indicated by high AMP and ADP levels), PFK-1 is activated, accelerating glycolysis to replenish ATP. When energy is abundant (high ATP, citrate levels), PFK-1 is inhibited, slowing glucose consumption. Low pH also inhibits PFK-1, protecting cells from excessive acid production during anaerobic glycolysis. The most potent activator of PFK-1 is fructose-2,6-bisphosphate (F-2,6-BP), a regulatory molecule (not a glycolytic intermediate) whose concentration is controlled by hormonal signals. This mechanism links glycolytic rate to the fed/fasted state of the organism. Panel A: PFK-1 tetrameric structure with active site in core and allosteric regulatory sites on surface. Panel B: Activators (green arrows) including AMP, ADP, fructose-2,6-bisphosphate, and Pi pointing to regulatory sites. Panel C: Inhibitors (red arrows) including ATP, citrate, and H+ indicating low pH effects. Panel D: T state to R state conformational shift with sigmoidal velocity curve showing activation leftward shift and inhibition rightward shift. #### Step 4: Aldolase Aldolase catalyzes the reversible cleavage of the six-carbon fructose-1,6-bisphosphate into two three-carbon molecules: dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This is the "splitting" that gives glycolysis its name. Although the equilibrium favors the condensation direction (reforming fructose-1,6-bisphosphate), the continuous removal of products by subsequent reactions pulls the reaction forward. #### Step 5: Triose Phosphate Isomerase Only glyceraldehyde-3-phosphate can proceed through the payoff phase of glycolysis. Triose phosphate isomerase rapidly interconverts the two triose phosphates, converting all DHAP to G3P. This enzyme approaches catalytic perfection—its kcat/Km ratio is so high that the reaction rate is limited only by how fast substrates can diffuse to the enzyme. From this point forward, every reaction occurs twice per original glucose molecule. Panel A: Glucose entering pathway with hexokinase step 1 (ATP to ADP) producing G6P and side panel comparing hexokinase versus glucokinase properties. Panel B: Phosphoglucose isomerase step 2 converting G6P to F6P and PFK-1 step 3 (red box, rate-limiting) producing F1,6BP. Panel C: Aldolase step 4 cleaving F1,6BP into DHAP and G3P shown as 3-carbon structures. Panel D: Triose phosphate isomerase step 5 converting DHAP to G3P with reversible equilibrium arrow and net -2 ATP tally. --- ### III. Payoff Phase (Steps 6-10) The payoff phase extracts energy from glyceraldehyde-3-phosphate, generating NADH and ATP. Because each glucose molecule produces two G3P molecules, all reactions in this phase occur twice per glucose. #### Step 6: Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) This reaction couples an oxidation with a phosphorylation, capturing energy that would otherwise be lost. Glyceraldehyde-3-phosphate is oxidized by NAD⁺ to form a carboxylic acid, but the enzyme immediately couples this to phosphorylation by inorganic phosphate (not ATP), generating 1,3-bisphosphoglycerate. The newly created acyl phosphate bond stores sufficient energy to drive ATP synthesis in the next step. This reaction produces the first NADH of glycolysis (two per glucose). The NADH must eventually be reoxidized to NAD⁺ for glycolysis to continue—how this occurs depends critically on whether oxygen is available. #### Step 7: Phosphoglycerate Kinase The high-energy acyl phosphate group of 1,3-bisphosphoglycerate is transferred to ADP, generating ATP and 3-phosphoglycerate. This is the first of two substrate-level phosphorylation reactions in glycolysis, producing ATP directly without the electron transport chain. This step yields two ATP per glucose, exactly recouping the two ATP invested in the preparatory phase. #### Step 8: Phosphoglycerate Mutase The phosphate group is repositioned from carbon 3 to carbon 2, converting 3-phosphoglycerate to 2-phosphoglycerate. This intramolecular rearrangement prepares the molecule for the next step by positioning the phosphate adjacent to a hydroxyl group that will be eliminated. #### Step 9: Enolase Enolase removes water from 2-phosphoglycerate, generating phosphoenolpyruvate (PEP). This dehydration dramatically increases the energy of the phosphate bond. While 2-phosphoglycerate contains a relatively low-energy phosphoester bond, phosphoenolpyruvate contains an extremely high-energy phosphate bond—high enough to drive ATP synthesis. Enolase requires magnesium ions for activity and is inhibited by fluoride ions. This property has clinical importance: fluoride is added to blood collection tubes for glucose measurements because inhibiting enolase stops glycolysis and prevents red blood cells from consuming glucose during sample processing. #### Step 10: Pyruvate Kinase The final step of glycolysis transfers the high-energy phosphate from phosphoenolpyruvate to ADP, generating ATP and pyruvate. This irreversible reaction (ΔG = −31.4 kJ/mol) represents the second substrate-level phosphorylation and produces two more ATP per glucose—the net profit of the pathway. Pyruvate kinase is the third regulatory enzyme of glycolysis. It is activated by fructose-1,6-bisphosphate in a feedforward mechanism: when PFK-1 is active and producing fructose-1,6-bisphosphate, pyruvate kinase activity increases to handle the increased flux. In liver, glucagon inhibits pyruvate kinase through phosphorylation, helping to prevent futile cycling when gluconeogenesis should predominate. Panel A: GAPDH step 6 with two G3P molecules (x2) producing NADH and 1,3-bisphosphoglycerate with high-energy acyl phosphate bond in yellow. Panel B: Phosphoglycerate kinase step 7 producing ATP and 3-phosphoglycerate (first substrate-level phosphorylation in green callout). Panel C: Phosphoglycerate mutase step 8 and enolase step 9 removing H2O to produce phosphoenolpyruvate with high-energy bond highlighted. Panel D: Pyruvate kinase step 10 (red regulation box) producing ATP and pyruvate with total yield summary +4 ATP, +2 NADH per glucose. --- ### IV. Energy Yield of Glycolysis The direct ATP yield from glycolysis is straightforward: two ATP are invested in the preparatory phase and four ATP are produced in the payoff phase, yielding a net gain of two ATP per glucose through substrate-level phosphorylation. This modest yield represents only a fraction of the energy stored in glucose—the complete oxidation of glucose through the citric acid cycle and oxidative phosphorylation yields approximately 30-32 ATP. The two NADH molecules produced per glucose represent additional potential energy. Under aerobic conditions, these electrons can be transferred into mitochondria through shuttle systems and fed into the electron transport chain. The malate-aspartate shuttle (found in heart, liver, and kidneys) preserves the electrons at the NADH level, yielding approximately 2.5 ATP per cytoplasmic NADH. The glycerol-3-phosphate shuttle (prominent in skeletal muscle and brain) transfers electrons at the FADH₂ level, yielding approximately 1.5 ATP per cytoplasmic NADH. Under anaerobic conditions, the NADH cannot be oxidized through the electron transport chain. Instead, it must be reoxidized in the cytoplasm to regenerate NAD⁺ and allow glycolysis to continue. This is accomplished through lactate dehydrogenase, which reduces pyruvate to lactate while oxidizing NADH back to NAD⁺. Although this sacrifices the energy that could have been captured from NADH oxidation, it permits continued ATP production under hypoxic conditions. Panel A: Aerobic conditions with pyruvate entering mitochondrion through pyruvate dehydrogenase to acetyl-CoA and TCA cycle. Panel B: NADH shuttle pathways showing malate-aspartate (liver, heart, 2.5 ATP) and glycerol-3-phosphate (muscle, brain, 1.5 ATP) with ETC producing ATP. Panel C: Anaerobic conditions with lactate dehydrogenase reducing pyruvate to lactate while regenerating NAD+ in cytoplasm. Panel D: Energy summaries comparing ~32 ATP aerobic total versus 2 ATP anaerobic with tissue examples (RBCs, exercising muscle, tumor cells). --- ### V. Fate of Pyruvate Pyruvate stands at a metabolic crossroads. Its fate depends on the cell's oxygen availability, metabolic needs, and enzymatic complement. #### Aerobic Metabolism: Entry into the Citric Acid Cycle When oxygen is available, pyruvate enters the mitochondria through a specific transporter and undergoes oxidative decarboxylation by the pyruvate dehydrogenase complex. This multi-enzyme complex removes carbon dioxide and transfers the remaining two-carbon fragment to coenzyme A, generating acetyl-CoA and NADH. Acetyl-CoA then enters the citric acid cycle, where its carbons are completely oxidized to carbon dioxide while generating additional NADH and FADH₂ for oxidative phosphorylation. This pathway extracts the maximum possible energy from glucose. #### Anaerobic Metabolism: Lactate Formation When oxygen is limiting or absent, the cell cannot reoxidize NADH through the electron transport chain. Glycolysis would quickly halt as NAD⁺ becomes depleted. Lactate dehydrogenase provides the solution by reducing pyruvate to lactate, regenerating NAD⁺ in the process: Pyruvate + NADH + H⁺ → Lactate + NAD⁺. This anaerobic pathway operates continuously in red blood cells, which lack mitochondria and depend entirely on glycolysis for ATP. During intense exercise, skeletal muscle produces lactate when oxygen delivery cannot keep pace with energy demands—the familiar sensation of "burning" muscles reflects lactate accumulation. Cancer cells often preferentially reduce pyruvate to lactate even when oxygen is available (the Warburg effect), a metabolic reprogramming that supports rapid cell proliferation. #### Gluconeogenesis: Glucose Regeneration In liver (and to a lesser extent kidney), pyruvate can be converted back to glucose through gluconeogenesis—essentially glycolysis in reverse with enzymes that bypass the three irreversible steps. The Cori cycle coordinates muscle and liver metabolism: lactate produced by working muscle travels through the bloodstream to the liver, where it is converted to pyruvate and then to glucose. This glucose returns to muscle, completing a cycle that effectively transfers the metabolic burden of regenerating glucose from NAD⁺-limited muscle to the aerobic liver. Panel A: Pyruvate structure (CH3-CO-COO-) at decision point with aerobic pathway showing entry to mitochondrion and pyruvate dehydrogenase conversion to acetyl-CoA. Panel B: TCA cycle wheel producing NADH, FADH2, CO2 with final yield ~30-32 ATP and CO2 + H2O. Panel C: Anaerobic fermentation pathway with lactate dehydrogenase converting pyruvate to lactate coupled to NADH to NAD+ regeneration. Panel D: Gluconeogenesis in liver converting pyruvate back to glucose with Cori cycle showing lactate from muscle to liver and glucose returning. --- ### VI. Regulation of Glycolysis Glycolytic flux is regulated primarily at the three irreversible steps, each catalyzed by an allosteric enzyme responsive to cellular energy status and hormonal signals. #### The Three Regulatory Enzymes Hexokinase (and glucokinase) controls entry of glucose into metabolism. Phosphofructokinase-1 (PFK-1) represents the most important regulatory point, controlling commitment to glycolysis. Pyruvate kinase controls exit from the pathway and final ATP production. #### Hormonal Regulation in Liver Hormones coordinate glycolytic activity with the body's overall metabolic state. In the fed state, insulin signals fuel abundance. Insulin promotes glycolysis by inducing glucokinase expression, activating PFK-2 (which increases fructose-2,6-bisphosphate), and dephosphorylating pyruvate kinase to activate it. In the fasted state, glucagon signals the need to produce and release glucose for other tissues. Glucagon inhibits glycolysis by inhibiting PFK-2 (decreasing fructose-2,6-bisphosphate), phosphorylating pyruvate kinase to inhibit it, and promoting gluconeogenesis through altered gene expression and enzyme phosphorylation. #### The Fructose-2,6-Bisphosphate Switch Fructose-2,6-bisphosphate (F-2,6-BP) is not a glycolytic intermediate but a regulatory signal molecule that potently activates PFK-1 and inhibits the gluconeogenic enzyme fructose-1,6-bisphosphatase. Its concentration is controlled by a remarkable bifunctional enzyme that possesses both kinase activity (PFK-2, which makes F-2,6-BP) and phosphatase activity (FBPase-2, which degrades F-2,6-BP). Glucagon-triggered phosphorylation of this bifunctional enzyme inhibits its kinase activity while activating its phosphatase activity, lowering F-2,6-BP levels and thereby inhibiting glycolysis while favoring gluconeogenesis. Insulin reverses this, promoting dephosphorylation and raising F-2,6-BP levels to stimulate glycolysis. This elegant mechanism coordinates glycolysis and gluconeogenesis in response to feeding state. Panel A: Bifunctional PFK-2/FBPase-2 enzyme with kinase domain (green) and phosphatase domain (red) interconverting F-6-P and F-2,6-BP. Panel B: Insulin (blue) promoting dephosphorylation activating kinase domain resulting in high F-2,6-BP and activated glycolysis. Panel C: Glucagon (orange) promoting phosphorylation via PKA inhibiting kinase domain resulting in low F-2,6-BP and favored gluconeogenesis. Panel D: F-2,6-BP as potent PFK-1 activator (green plus) and fructose-1,6-bisphosphatase inhibitor (red minus) switching metabolism. --- ### VII. Tissue-Specific Considerations Different tissues have adapted glycolysis to their specific needs through expression of tissue-specific isoenzymes and unique regulatory mechanisms. #### Red Blood Cells Mature red blood cells lack mitochondria, nucleus, and other organelles, relying entirely on glycolysis for ATP production. All glucose is converted to lactate, and the 2 ATP per glucose must sustain the membrane pumps that maintain the biconcave shape and ion gradients. Red blood cells also produce 2,3-bisphosphoglycerate (2,3-BPG) through a bypass of glycolysis; this molecule binds to hemoglobin and reduces its oxygen affinity, facilitating oxygen unloading in tissues. #### Brain The brain demands a continuous, substantial glucose supply—approximately 120 grams per day, representing about 20% of total body glucose consumption. Brain tissue cannot oxidize fatty acids because they do not cross the blood-brain barrier efficiently, making the brain critically dependent on blood glucose. During prolonged fasting, the brain adapts partially to ketone body use, but glucose remains essential. #### Skeletal Muscle Skeletal muscle has high glycolytic capacity, particularly in fast-twitch (type II) fibers designed for rapid, powerful contractions. During intense exercise exceeding oxygen delivery capacity, muscle produces large quantities of lactate. This lactate travels to the liver (Cori cycle) or heart (which preferentially oxidizes lactate) for further metabolism. #### Liver The liver uses glucokinase rather than hexokinase, enabling it to efficiently extract glucose from portal blood after meals while not trapping glucose during fasting states. Liver glycolysis is tightly regulated by hormones and can be reversed (gluconeogenesis) to produce glucose for export to other tissues. The liver is the primary site of the Cori cycle, converting lactate from muscle and red blood cells back to glucose. --- ### VIII. Clinical Correlations #### Pyruvate Kinase Deficiency Pyruvate kinase deficiency is the most common enzyme deficiency affecting glycolysis and causes chronic hemolytic anemia. Red blood cells, entirely dependent on glycolysis, cannot produce sufficient ATP to maintain membrane integrity and ion gradients when pyruvate kinase activity is reduced. The cells become rigid and are removed by the spleen. Interestingly, 2,3-BPG levels are elevated because precursors accumulate upstream of the block, leading to a right-shifted oxygen-hemoglobin dissociation curve that partially compensates for anemia by increasing oxygen delivery to tissues. #### Lactic Acidosis Lactic acidosis results from excessive lactate production or impaired lactate clearance. Tissue hypoxia (from shock, sepsis, or severe hypoxemia) forces tissues to rely on anaerobic glycolysis, generating large amounts of lactate. Thiamine (vitamin B1) deficiency impairs pyruvate dehydrogenase, blocking pyruvate entry into the citric acid cycle and shunting it toward lactate. Metformin toxicity, particularly in patients with renal impairment, can cause severe lactic acidosis by inhibiting hepatic gluconeogenesis and cellular respiration. Inherited mitochondrial disorders also cause lactic acidosis by impairing oxidative phosphorylation. #### The Warburg Effect Otto Warburg observed in the 1920s that tumor cells consume glucose at high rates and produce lactate even in the presence of adequate oxygen—a phenomenon now called aerobic glycolysis or the Warburg effect. While seemingly wasteful (yielding only 2 ATP versus approximately 32 from complete oxidation), this metabolic reprogramming provides advantages for rapidly proliferating cells: glycolytic intermediates supply biosynthetic pathways for nucleotides, amino acids, and lipids needed to construct new cells. The Warburg effect forms the basis for positron emission tomography (PET) imaging with fluorodeoxyglucose (¹⁸F-FDG). This glucose analog is taken up by cells via glucose transporters and phosphorylated by hexokinase but cannot proceed further in glycolysis. It accumulates in cells with high glycolytic rates, including most cancers. PET scanning detects this accumulation, enabling tumor visualization, staging, and monitoring of treatment response. Panel A: Normal cell metabolism with glucose fully oxidized through glycolysis, TCA cycle, and oxidative phosphorylation producing ~32 ATP. Panel B: Cancer cell with increased glucose transporters converting glucose to lactate (aerobic glycolysis) producing 2 ATP plus biosynthetic precursors. Panel C: 18F-FDG PET principle showing FDG entering via GLUT, phosphorylation by hexokinase to trapped FDG-6-phosphate emitting positrons. Panel D: Whole-body PET scan showing normal brain and bladder uptake with abnormal tumor focus and SUV color scale from blue to red. --- ## Summary Glycolysis is the universal cytoplasmic pathway that converts one molecule of glucose to two molecules of pyruvate through ten enzymatic steps. The investment phase (steps 1-5) consumes two ATP to phosphorylate and cleave glucose into two triose phosphates. The payoff phase (steps 6-10) generates four ATP and two NADH, yielding a net gain of two ATP per glucose. Three irreversible enzymes—hexokinase/glucokinase, phosphofructokinase-1, and pyruvate kinase—serve as regulatory points. PFK-1 is the rate-limiting enzyme, activated by low energy signals (AMP, ADP) and the potent activator fructose-2,6-bisphosphate, while being inhibited by high energy indicators (ATP, citrate). Pyruvate's fate depends on oxygen availability: under aerobic conditions, it enters mitochondria to be completely oxidized; under anaerobic conditions, it is reduced to lactate to regenerate NAD⁺. Tissue-specific adaptations include the use of glucokinase in liver, complete dependence on glycolysis in red blood cells, and high glycolytic capacity in skeletal muscle. Clinical conditions related to glycolysis include pyruvate kinase deficiency (hemolytic anemia), lactic acidosis, and the Warburg effect in cancer. --- ## Key Terms | Term | Definition | |------|------------| | Glycolysis | Ten-step cytoplasmic pathway converting glucose to pyruvate with net production of 2 ATP and 2 NADH | | Substrate-level phosphorylation | Direct transfer of a phosphate group from a high-energy substrate to ADP, producing ATP | | Phosphofructokinase-1 (PFK-1) | Rate-limiting enzyme of glycolysis that phosphorylates fructose-6-phosphate; allosterically regulated by energy status | | Fructose-2,6-bisphosphate | Regulatory molecule and most potent activator of PFK-1; controlled by the bifunctional enzyme PFK-2/FBPase-2 | | Lactate fermentation | Anaerobic pathway regenerating NAD⁺ by reducing pyruvate to lactate via lactate dehydrogenase | | Warburg effect | Preferential use of glycolysis by cancer cells even in the presence of oxygen; basis for FDG-PET imaging | | Cori cycle | Metabolic cycle where lactate from muscle travels to liver for conversion back to glucose | --- *This content is subject to the [MIT License](https://opensource.org/licenses/MIT). © 2024–2026 Hibbert School of Medicine.*