Original episode & show notes | Raw transcript
Glycolysis, derived from the Greek words glycos (sweet) and lysis (to split), is the foundational metabolic pathway for breaking down glucose. It is a sequence of ten enzyme-catalyzed reactions that converts a single molecule of glucose into two molecules of pyruvate. This process serves two primary functions: to generate ATP, the cell’s immediate energy currency, and to produce intermediates that can be used in other metabolic pathways. As we’ll explore, this pathway is a masterclass in biochemical efficiency, regulation, and integration, operating in the cytosol (the main fluid-filled space) of every cell.
Cells cannot store large quantities of ATP directly. ATP is an energetically “expensive” molecule to maintain because the reaction that breaks it down (ATP → ADP + Pᵢ) is held far from its natural equilibrium. This disequilibrium is what gives ATP its high energy-releasing potential. Think of it like a compressed spring; its energy comes from its state of high tension, not the material itself.
Instead of storing massive amounts of this “ready cash,” the cell stores energy more stably in the form of carbohydrates (like glycogen) and fats. Glycolysis is the first step in unlocking the energy stored in glucose, acting like a pre-filled form that can be quickly “signed” to release energy when needed.
Every step in glycolysis is facilitated by a specific enzyme. To understand metabolism, it’s crucial to understand how enzymes work.
Lowering Activation Energy: Chemical reactions require a certain amount of energy to get started, known as the activation energy. Imagine two puddles of water separated by a hill. For water to move from the higher puddle to the lower one, it needs enough energy to be “kicked” over the top of the hill. An enzyme acts by lowering the height of this hill, making it vastly more likely for molecules (which are constantly bouncing around with varying energy levels) to make it over to the other side.
Reversible vs. Irreversible Reactions:
Equilibrium Reactions: Many enzymes can catalyze reactions in both the forward and reverse directions. In our analogy, the hill is low enough that molecules can be kicked over from either side. The reaction reaches equilibrium, where the rate of the forward reaction equals the rate of thereverse reaction. This does not mean the amount of reactants and products is equal. For example, the conversion of pyruvate to lactate via the enzyme lactate dehydrogenase (LDH) heavily favors lactate, with a ratio of about 10:1 at rest and 100:1 during intense exercise.
Irreversible Reactions: Some reactions have a very large energy drop, like water flowing down a massive waterfall. The “hill” to go in the reverse direction is simply too high for molecules to overcome. These steps are effectively one-way streets and are the key points where the entire pathway is regulated.
Rate-Limiting Steps: Not all enzymes work at the same speed. The slowest enzyme in a pathway acts as a bottleneck, determining the overall speed (or flux) of the entire process. These are known as rate-limiting steps and are often the same irreversible steps that are heavily regulated.
Glycolysis is a ten-step process, logically divided into two distinct phases.
This phase prepares the six-carbon glucose molecule for splitting by investing two molecules of ATP. The goal is to create two identical, high-energy three-carbon molecules.
First Investment (Phosphorylation): Glucose enters the cell and is immediately phosphorylated by the enzyme hexokinase. A phosphate group from an ATP molecule is attached to the 6th carbon of glucose, forming glucose-6-phosphate (G6P). This step costs 1 ATP. This phosphorylation traps glucose inside the cell and destabilizes it for the next reaction. This is one of the key irreversible, regulatory steps.
Isomerization: The G6P is converted into its isomer, fructose-6-phosphate (F6P). An isomer has the same chemical formula but a different atomic arrangement. This rearrangement from a six-membered ring to a five-membered ring is crucial for the next step.
Second Investment (The Key Regulatory Step): Another ATP molecule is invested. The enzyme phosphofructokinase (PFK) adds a second phosphate group, creating fructose-1,6-bisphosphate (F1,6BP). This costs another 1 ATP. This is the most important rate-limiting and irreversible step in all of glycolysis. When the cell has plenty of energy (high ATP), PFK is inhibited, slowing glycolysis down. When the cell needs energy, PFK is activated.
The Split: The enzyme aldolase splits the six-carbon F1,6BP molecule directly in half, yielding two different three-carbon sugar phosphates: glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).
Final Isomerization: DHAP is rapidly converted into a second molecule of GAP.
End of Phase 1 Summary: We started with one glucose molecule and invested 2 ATP. We now have two identical molecules of GAP, ready for the energy payoff.
In this phase, the two GAP molecules are converted to pyruvate. This process generates ATP and NADH. Remember, every reaction from this point forward happens twice for each starting glucose molecule.
Oxidation and Phosphorylation: Each GAP molecule is oxidized. In this process, a hydrogen and its electrons are transferred to the coenzyme NAD⁺, forming NADH. Simultaneously, an inorganic phosphate from the cytosol is attached, creating 1,3-bisphosphoglycerate (1,3-BPG). This molecule is a very high-energy intermediate. This step produces 2 NADH total (one for each GAP).
First ATP Payoff: The high-energy phosphate on 1,3-BPG is transferred directly to an ADP molecule, forming ATP. This is called substrate-level phosphorylation. This reaction is so favorable that it pulls the previous, less favorable reaction (Step 6) forward in what is known as a coupled reaction. This step produces 2 ATP total. We are now left with 3-phosphoglycerate.
Phosphate Shuffle: The phosphate group is moved from the 3rd carbon to the 2nd carbon, forming 2-phosphoglycerate.
Creating a High-Energy Intermediate: A molecule of water is removed, creating a double bond. This transforms the molecule into phosphoenolpyruvate (PEP). PEP is an extremely high-energy, unstable molecule, primed to release its phosphate group.
Final ATP Payoff: The phosphate group from PEP is transferred to ADP, forming our final ATP. This is the third and final irreversible step of glycolysis. The reaction is so energetically favorable that it’s a massive “waterfall.” This step produces 2 ATP total and leaves us with the final product: pyruvate.
End of Phase 2 Summary: From the two GAP molecules, we generated a total of 4 ATP and 2 NADH.
For one molecule of glucose, the net production is:
2 Pyruvate molecules
2 ATP molecules (4 produced - 2 invested)
2 NADH molecules
Step 6 of glycolysis requires the coenzyme NAD⁺. If glycolysis runs continuously, all the cell’s NAD⁺ will be converted to NADH, and the pathway would grind to a halt. The cell must regenerate NAD⁺ from NADH for glycolysis to continue. This is where the fate of pyruvate becomes critical, especially in the absence of oxygen (anaerobic conditions).
Lactic Acid Fermentation (In Humans): During intense exercise, when oxygen is limited, cells regenerate NAD⁺ by transferring the hydrogen from NADH directly onto pyruvate. This reaction, catalyzed by lactate dehydrogenase, converts pyruvate into lactate. This is why lactate levels rise during hard efforts—it’s a direct consequence of rapid, anaerobic glycolysis. The process is a cycle: glycolysis produces NADH, and lactate formation consumes it, allowing energy production to continue.
Alcoholic Fermentation (In Yeast): Yeast and some other microorganisms use a different method. First, they remove a carbon from pyruvate, releasing it as CO₂. This leaves a two-carbon molecule called acetaldehyde. Then, to regenerate NAD⁺, they transfer the hydrogen from NADH to acetaldehyde, producing ethanol (drinking alcohol).
Anaerobic Energy Production: Glycolysis does not directly use oxygen. This makes it the primary system for generating ATP very quickly during short, intense bursts of effort (e.g., sprinting, lifting heavy weights) when the oxygen-dependent aerobic system cannot keep up with demand.
Location, Location, Location: The fact that glycolysis occurs in the cytosol, not the mitochondria, is key to its speed. It’s an immediate, on-demand energy source available right where it’s needed.
A Central Metabolic Hub: Glycolysis is not an isolated pathway. Its intermediates are siphoned off to build other essential molecules (like amino acids and nucleic acids), and it serves as the entry point for other sugars (like fructose) into metabolism. The final product, pyruvate, stands at a critical crossroads: it can be converted to lactate (anaerobically) or it can be shuttled into the mitochondria to be fully oxidized for much greater ATP yield (aerobically), a topic for another day.