Glycolysis Steps

Why This Matters

Glycolysis isn't just a list of ten reactions to memorize—it's the universal energy-extraction pathway that every living cell uses, from bacteria to your brain cells. You're being tested on your understanding of substrate-level phosphorylation, enzyme regulation, and energy investment versus payoff. The pathway demonstrates core biochemical principles: how cells trap metabolites, commit to irreversible pathways, and couple unfavorable reactions to favorable ones.

When you encounter glycolysis on an exam, you need to recognize which steps consume ATP versus produce it, identify the key regulatory enzymes, and explain why certain reactions are irreversible. Don't just memorize the sequence—know what concept each step illustrates and how the pathway's logic reflects cellular energy management.

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Energy Investment Phase: Priming the Fuel

The first half of glycolysis requires ATP input to destabilize glucose and prepare it for cleavage. This investment phase uses 2 ATP per glucose molecule, creating a phosphorylated, committed substrate that cannot escape the cell.

Step 1: Glucose Phosphorylation (Hexokinase)

• Hexokinase traps glucose—adding a phosphate from ATP creates glucose-6-phosphate, which cannot cross the plasma membrane
• ATP consumption begins here—this is the first of two ATP-spending steps in the investment phase
• Irreversible under cellular conditions—the large negative $\Delta G$ commits glucose to metabolism (though a different enzyme, glucose-6-phosphatase, reverses this in gluconeogenesis)

Step 2: Isomerization to Fructose-6-Phosphate (Phosphoglucose Isomerase)

• Structural rearrangement from aldose to ketose—converts the six-membered ring to a five-membered ring, repositioning the carbonyl group
• Reversible reaction—near-equilibrium step that allows metabolic flexibility between glycolysis and gluconeogenesis
• Prepares for symmetric cleavage—the ketose structure enables aldolase to split the molecule into two equivalent three-carbon units

Step 3: Committed Step (Phosphofructokinase-1)

• PFK-1 is the major regulatory enzyme—catalyzes phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate using ATP
• Irreversible and rate-limiting—this is the "point of no return" that commits the cell to completing glycolysis
• Allosteric regulation hub—inhibited by ATP and citrate (energy abundance), activated by AMP and fructose-2,6-bisphosphate (energy need)

Step 4: Cleavage (Aldolase)

• Aldolase splits the 6-carbon sugar—fructose-1,6-bisphosphate becomes dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P)
• Thermodynamically unfavorable but pulled forward—the reaction has positive $\Delta G°'$ but proceeds because products are rapidly consumed
• Only G3P continues directly—DHAP must be converted to G3P by triose phosphate isomerase, effectively doubling the pathway from this point

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Energy Payoff Phase: Harvesting ATP and NADH

The second half extracts energy from the two three-carbon molecules. Each G3P generates 2 ATP and 1 NADH, so one glucose yields 4 ATP and 2 NADH in this phase—a net gain of 2 ATP after subtracting the investment.

Step 5: Oxidation and Phosphorylation (Glyceraldehyde-3-Phosphate Dehydrogenase)

• First energy capture as NADH—G3P is oxidized while $NAD^+$ is reduced to $NADH$, storing electrons for later ATP production
• Inorganic phosphate added (not ATP)—creates the high-energy compound 1,3-bisphosphoglycerate with an acyl phosphate bond
• Couples oxidation to phosphorylation—the energy released from oxidation drives the thermodynamically unfavorable phosphate addition

Step 6–7: First ATP Generation (Phosphoglycerate Kinase)

• Substrate-level phosphorylation produces ATP—the high-energy phosphate from 1,3-bisphosphoglycerate transfers directly to ADP
• Two ATP generated per glucose—remember, two G3P molecules proceed through this step from one glucose
• Product is 3-phosphoglycerate—the phosphate remains on carbon 3 after the energy-rich acyl phosphate is donated

Step 8: Phosphate Migration (Phosphoglycerate Mutase)

• Mutase shifts phosphate from C3 to C2—converts 3-phosphoglycerate to 2-phosphoglycerate
• Requires a phosphorylated enzyme intermediate—the mechanism involves transient 2,3-bisphosphoglycerate formation
• Sets up dehydration—positioning the phosphate adjacent to the hydroxyl group enables the next step's water removal

Step 9: Dehydration (Enolase)

• Water removal creates a high-energy bond—enolase converts 2-phosphoglycerate to phosphoenolpyruvate (PEP)
• PEP has the highest phosphate transfer potential in glycolysis—the enol phosphate bond stores significant energy due to tautomerization after hydrolysis
• Inhibited by fluoride—this is why fluoride is added to blood collection tubes to prevent glycolysis from depleting glucose

Step 10: Final ATP Generation (Pyruvate Kinase)

• Second substrate-level phosphorylation—PEP donates its phosphate to ADP, producing ATP and pyruvate
• Irreversible reaction—large negative $\Delta G$ makes this a regulatory point (pyruvate kinase is activated by fructose-1,6-bisphosphate)
• Pyruvate's fate depends on oxygen—enters the citric acid cycle aerobically or is reduced to lactate (or ethanol in yeast) anaerobically to regenerate $NAD^+$

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Regulatory Control Points

Glycolysis is controlled at three irreversible steps where dedicated enzymes prevent wasteful cycling between glycolysis and gluconeogenesis. These checkpoints respond to cellular energy status, ensuring glucose is metabolized only when ATP is needed.

Hexokinase Regulation

• Product inhibition by glucose-6-phosphate—prevents ATP waste when downstream metabolism is saturated
• Tissue-specific isozymes matter—liver glucokinase has higher $K_m$ and isn't inhibited by product, allowing glucose storage as glycogen
• Trapping function is primary—regulation here controls glucose uptake rather than glycolytic flux

PFK-1 Regulation (Primary Control Point)

• ATP is both substrate and inhibitor—high ATP signals energy sufficiency and slows glycolysis despite being required for the reaction
• Fructose-2,6-bisphosphate is the most potent activator—this regulatory molecule (not a glycolytic intermediate) overrides ATP inhibition
• Citrate inhibition links glycolysis to the citric acid cycle—abundant citrate indicates sufficient biosynthetic precursors

Pyruvate Kinase Regulation

• Feed-forward activation by fructose-1,6-bisphosphate—ensures the payoff phase keeps pace with the investment phase
• Inhibited by ATP and alanine—alanine signals amino acid abundance (pyruvate is alanine's precursor)
• Hormonal control via phosphorylation—glucagon triggers phosphorylation that inactivates liver pyruvate kinase, sparing glucose for other tissues

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Quick Reference Table

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Self-Check Questions

1. Which two enzymes catalyze substrate-level phosphorylation, and what distinguishes their reactions thermodynamically?

2. Why is PFK-1 considered the committed step of glycolysis rather than hexokinase, even though both reactions are irreversible?

3. Compare the energy investment phase and payoff phase: how many ATP molecules are consumed versus produced, and what is the net ATP yield per glucose?

4. If a cell has high ATP and citrate levels, which glycolytic enzyme is most directly inhibited, and what is the physiological logic behind this regulation?

5. Explain why the conversion of PEP to pyruvate releases enough energy to drive ATP synthesis, referencing the concept of phosphate transfer potential. (FRQ-style: This tests your ability to connect thermodynamics to pathway logic.)