Glycolysis Step by Step

Glycolysis (from Greek, “sugar splitting”) is the ten-step pathway that breaks one molecule of glucose into two molecules of pyruvate. It runs in the cytoplasm of almost every living cell, needs no oxygen, and is one of the oldest metabolic pathways on Earth. It’s also a beautiful case study in organic chemistry: isomerisations, aldol reactions, oxidations and phosphate transfers, each catalysed by its own enzyme.

The overall picture

Glucose + 2 NAD⁺ + 2 ADP + 2 Pᵢ → 2 pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 H₂O

The pathway has two phases:

• Investment phase (steps 1–5): the cell spends 2 ATP to phosphorylate glucose and split it into two 3-carbon sugar phosphates.
• Payoff phase (steps 6–10): each 3-carbon unit is oxidised, producing NADH, and its phosphate groups are transferred to ADP, making 4 ATP in total.

Net: 2 ATP and 2 NADH per glucose.

Investment phase

Step 1: Glucose → glucose-6-phosphate

• Enzyme: hexokinase (glucokinase in the liver)
• Reaction: transfer of a phosphate group from ATP to the –OH on carbon 6.
• ATP: −1
• Why: the negatively charged phosphate traps glucose inside the cell (it can’t cross the membrane) and “activates” it for later steps. The reaction is strongly favourable because it’s coupled to ATP hydrolysis (see ATP: the cell’s energy currency).

Step 2: Glucose-6-phosphate → fructose-6-phosphate

• Enzyme: phosphoglucose isomerase
• Reaction: isomerisation of an aldose to a ketose (the carbonyl group moves from carbon 1 to carbon 2), via the open-chain form (see glucose: ring and chain forms).
• Why: puts the molecule into a form that can be phosphorylated on carbon 1 and later split symmetrically.

Step 3: Fructose-6-phosphate → fructose-1,6-bisphosphate

• Enzyme: phosphofructokinase-1 (PFK-1)
• Reaction: a second phosphate transferred from ATP, onto carbon 1.
• ATP: −1
• Why: this is the committed step of glycolysis and its main control point (see “Regulation” below). The product now has a phosphate at each end.

Step 4: Fructose-1,6-bisphosphate → DHAP + G3P

• Enzyme: aldolase
• Reaction: a reverse aldol reaction splits the six-carbon chain between carbons 3 and 4 into two three-carbon sugar phosphates:
  • dihydroxyacetone phosphate (DHAP), a ketone;
  • glyceraldehyde-3-phosphate (G3P), an aldehyde.
• Why: this is the “splitting” in glycolysis. Under standard conditions, the reaction is actually unfavourable, but in cells, products are removed quickly, which keeps it going.

Step 5: DHAP → G3P

• Enzyme: triose phosphate isomerase
• Reaction: isomerisation of the ketone DHAP into the aldehyde G3P.
• Why: only G3P continues in the pathway, so converting DHAP means both halves of the glucose molecule are used. Triose phosphate isomerase is one of the fastest known enzymes, working at close to the limit set by diffusion (see enzyme kinetics).

After step 5: one glucose has become two G3P molecules, at a cost of 2 ATP. Every following step happens twice per glucose.

Payoff phase (each step ×2)

Step 6: G3P → 1,3-bisphosphoglycerate

• Enzyme: glyceraldehyde-3-phosphate dehydrogenase
• Reaction: the aldehyde group is oxidised, and inorganic phosphate is added, forming a high-energy acyl phosphate at carbon 1.
• NAD⁺ → NADH: the electrons removed from G3P are passed to NAD⁺ as a hydride ion (see cofactors and coenzymes).
• Why: this is the only oxidation in glycolysis. The energy released by oxidising the aldehyde is captured in the high-energy phosphate bond, rather than lost as heat.

Step 7: 1,3-bisphosphoglycerate → 3-phosphoglycerate

• Enzyme: phosphoglycerate kinase
• Reaction: the high-energy phosphate on carbon 1 is transferred to ADP, making ATP.
• ATP: +1 (×2 = +2)
• Why: this is substrate-level phosphorylation: ATP made directly from a high-energy intermediate, without the electron transport chain. At this point, the 2 ATP invested have been “repaid”.

Step 8: 3-phosphoglycerate → 2-phosphoglycerate

• Enzyme: phosphoglycerate mutase
• Reaction: the phosphate group moves from carbon 3 to carbon 2.
• Why: sets up the next step.

Step 9: 2-phosphoglycerate → phosphoenolpyruvate (PEP)

• Enzyme: enolase (needs Mg²⁺)
• Reaction: dehydration: a water molecule is removed, forming a C=C double bond.
• Why: this rearrangement turns a low-energy phosphate ester into phosphoenolpyruvate, one of the highest-energy phosphate compounds in metabolism. The energy was always there; removing water redistributes it so it’s released on phosphate transfer. (Fluoride inhibits enolase, which is why sodium fluoride is added to blood samples for glucose testing: it stops blood cells using up the glucose.)

Step 10: PEP → pyruvate

• Enzyme: pyruvate kinase
• Reaction: the phosphate is transferred from PEP to ADP, making ATP. The resulting enol immediately rearranges (tautomerises) into the stable keto form, pyruvate, which makes the reaction strongly favourable.
• ATP: +1 (×2 = +2)

The balance sheet (per glucose)

	ATP used	ATP made	NADH made
Steps 1 and 3	2
Step 6 (×2)			2
Step 7 (×2)		2
Step 10 (×2)		2
Net		+2 ATP	+2 NADH

Regulation: keeping glycolysis in check

Three steps (1, 3 and 10) have large negative free energy changes in cells and are effectively irreversible. They’re the natural control points, and PFK-1 (step 3) is the most important:

• Inhibited by high ATP (“plenty of energy, slow down”) and by citrate (a sign the Krebs cycle is well supplied).
• Activated by AMP and ADP (“energy is running low, speed up”).

This is a clear example of allosteric regulation and feedback inhibition (see competitive vs non-competitive inhibition). Hexokinase is inhibited by its own product, glucose-6-phosphate, and pyruvate kinase is also regulated.

What happens to pyruvate?

The fate of pyruvate depends on conditions, and the key issue is regenerating NAD⁺, because glycolysis stops at step 6 without it.

• With oxygen: pyruvate enters the mitochondria, is converted to acetyl-CoA and fed into the Krebs cycle. NADH is reoxidised by the electron transport chain.
• Without oxygen, in animal muscle: pyruvate is reduced to lactate by lactate dehydrogenase, using NADH and regenerating NAD⁺.
• Without oxygen, in yeast: pyruvate loses CO₂ to form acetaldehyde, which is reduced to ethanol, regenerating NAD⁺. This is alcoholic fermentation, used in brewing and bread-making.

These pathways are compared in aerobic vs anaerobic respiration.

Why glycolysis matters

• It’s the only ATP source for red blood cells (which have no mitochondria) and a fast source for sprinting muscles.
• Many cancer cells rely heavily on glycolysis even when oxygen is available (the Warburg effect), which is why PET scans using a radioactive glucose analogue light up many tumours.
• Its intermediates feed into making amino acids, fats and other molecules.

Glycolysis in reverse: making glucose

Cells can also run a pathway that looks like glycolysis backwards. Gluconeogenesis (“making new glucose”) builds glucose from smaller molecules such as lactate, pyruvate, glycerol and some amino acids, mainly in the liver. Seven of the ten steps of glycolysis are simply reversed by the same enzymes. The three irreversible steps (1, 3 and 10) can’t be reversed directly, so the liver uses different enzymes to bypass them, spending extra ATP and GTP. Gluconeogenesis keeps blood glucose up during fasting and recycles the lactate produced by hard-working muscles, a loop known as the Cori cycle.

Key takeaways

• Glycolysis converts 1 glucose → 2 pyruvate in 10 enzyme-catalysed steps in the cytoplasm.
• Investment phase (steps 1–5) uses 2 ATP and splits glucose into two G3P.
• Payoff phase (steps 6–10) makes 4 ATP by substrate-level phosphorylation and 2 NADH from one oxidation step.
• Net yield: 2 ATP + 2 NADH, with PFK-1 as the key control point.
• Pyruvate goes on to the Krebs cycle with oxygen, or to lactate or ethanol without it. See the whole pathway in cellular respiration.