IMAT Biology · Bioenergetics and Metabolism

Glycolysis and the Link Reaction

In the cytoplasm, glucose is split into two pyruvate for a net 2 ATP and 2 NADH; in the mitochondrial matrix, each pyruvate then loses a CO₂ and becomes acetyl coenzyme A.

All Bioenergetics and Metabolism notes

Why this matters

Glycolysis is asked in detail: the ministry papers of 2023 and 2025 named its enzymes (which steps use an isomerase) and asked how many NAD⁺ it reduces. Older papers asked the order of its stages, where CO₂ is first released, and which products glycolysis and the link reaction share.

Concept 1 of 2: Glycolysis: the ten steps from glucose to pyruvate

Glycolysis splits one glucose (6 carbons) into two pyruvate (3 carbons each). It first spends 2 ATP to make glucose reactive and easy to split, then earns 4 ATP and 2 NADH as the two halves are oxidised. It needs no oxygen, so it runs in every cell, with or without oxygen.

Definition

Site: the cytoplasm (cytosol). It needs no oxygen and is the same in aerobic and anaerobic conditions.
Investment phase: glucose is phosphorylated twice, using 2 ATP, giving fructose 1,6-bisphosphate. This 6-carbon sugar splits into two triose phosphates (3 carbons each).
Payoff phase: each triose phosphate is oxidised, reducing one $\mathrm{NAD^+}$ , and gives 2 ATP by substrate-level phosphorylation.
Net per glucose: 2 pyruvate, 2 ATP, 2 NADH. No $\mathrm{CO_2}$ is released and no $\mathrm{FADH_2}$ is made.
Enzyme families: kinases move a phosphate (to or from ATP), isomerases rearrange a molecule without adding anything, a dehydrogenase removes hydrogen (reducing $\mathrm{NAD^+}$ ). Phosphofructokinase is the main control point.

Step	Change	Enzyme	ATP or NADH
1	Glucose → glucose 6-phosphate	Hexokinase	1 ATP used
2	Glucose 6-phosphate → fructose 6-phosphate	Phosphoglucose isomerase	None
3	Fructose 6-phosphate → fructose 1,6-bisphosphate	Phosphofructokinase	1 ATP used
4	Fructose 1,6-bisphosphate → dihydroxyacetone phosphate + glyceraldehyde 3-phosphate	Aldolase	None
5	Dihydroxyacetone phosphate → glyceraldehyde 3-phosphate	Triose phosphate isomerase	None
6	Glyceraldehyde 3-phosphate → 1,3-bisphosphoglycerate	Glyceraldehyde 3-phosphate dehydrogenase	1 NADH made
7	1,3-bisphosphoglycerate → 3-phosphoglycerate	Phosphoglycerate kinase	1 ATP made
8	3-phosphoglycerate → 2-phosphoglycerate	Phosphoglycerate mutase	None
9	2-phosphoglycerate → phosphoenolpyruvate (water removed)	Enolase	None
10	Phosphoenolpyruvate → pyruvate	Pyruvate kinase	1 ATP made

Steps 6 to 10 happen twice per glucose, once for each triose phosphate: 2 NADH and 4 ATP made, minus 2 ATP used in steps 1 and 3.

Practice this conceptself-check · 4 quick reps

Gross is not net

Glycolysis makes 4 ATP per glucose but spends 2 in the investment phase, so the net yield is 2 ATP. Options with 4 ATP give the gross figure.

Glycolysis releases no CO₂

Glucose has 6 carbons and the two pyruvates have 3 each, so no carbon is lost. In aerobic respiration the first

\mathrm{CO_2}

is released in the link reaction; in yeast, the fermentation step after glycolysis releases it.

Isomerases rearrange; kinases phosphorylate

The isomerase steps change one sugar phosphate into another with the same atoms: glucose 6-phosphate to fructose 6-phosphate, and dihydroxyacetone phosphate to glyceraldehyde 3-phosphate. Adding a phosphate from ATP is done by a kinase, and splitting the 6-carbon sugar by aldolase.

Concept 2 of 2: The link reaction: pyruvate to acetyl coenzyme A

Pyruvate cannot enter the Krebs cycle as it is. Inside the mitochondrion it loses one carbon as

\mathrm{CO_2}

and two electrons to

\mathrm{NAD^+}

, and the 2-carbon acetyl group left over is attached to coenzyme A. Acetyl coenzyme A is the common entry point to the Krebs cycle for carbohydrate, fat and some amino acids.

Definition

Pyruvate is carried from the cytoplasm into the mitochondrial matrix.
The pyruvate dehydrogenase complex carries out an oxidative decarboxylation:
$\text{pyruvate (3C)} + \text{CoA} + \mathrm{NAD^+} \rightarrow \text{acetyl CoA (2C)} + \mathrm{CO_2} + \mathrm{NADH}$
Per glucose (two pyruvates): 2 acetyl CoA, 2 $\mathrm{CO_2}$ , 2 NADH, and no ATP.
Oxygen is not used in this step. It is needed later, at the end of the respiratory chain, to reoxidise the NADH; without it the link reaction soon stops.
Acetyl CoA is also made by the breakdown of fatty acids (β-oxidation) and of some amino acids.

Product	Per pyruvate	Per glucose	What happens to it
Acetyl CoA	1	2	Enters the Krebs cycle
CO₂	1	2	Diffuses out; the first CO₂ of aerobic respiration
NADH	1	2	Reoxidised by the respiratory chain
ATP	0	0	No ATP is made in this step

Practice this conceptself-check · 4 quick reps

Decarboxylation releases CO₂ but uses no oxygen

In the link reaction the

\mathrm{CO_2}

comes from pyruvate's own carboxyl group, not from oxygen gas. Oxygen gas is used only at the end of the respiratory chain, where it accepts electrons and forms water.

Summary — formulas & gotchas at a glance

A revision cheat-sheet for the formulas and gotchas above. Click any concept name to jump back to its full explanation.

Reference tables (2)

Glycolysis: the ten steps from glucose to pyruvate10 rows

Step	Change	Enzyme	ATP or NADH
1	Glucose → glucose 6-phosphate	Hexokinase	1 ATP used
2	Glucose 6-phosphate → fructose 6-phosphate	Phosphoglucose isomerase	None
3	Fructose 6-phosphate → fructose 1,6-bisphosphate	Phosphofructokinase	1 ATP used
4	Fructose 1,6-bisphosphate → dihydroxyacetone phosphate + glyceraldehyde 3-phosphate	Aldolase	None
5	Dihydroxyacetone phosphate → glyceraldehyde 3-phosphate	Triose phosphate isomerase	None
6	Glyceraldehyde 3-phosphate → 1,3-bisphosphoglycerate	Glyceraldehyde 3-phosphate dehydrogenase	1 NADH made
7	1,3-bisphosphoglycerate → 3-phosphoglycerate	Phosphoglycerate kinase	1 ATP made
8	3-phosphoglycerate → 2-phosphoglycerate	Phosphoglycerate mutase	None
9	2-phosphoglycerate → phosphoenolpyruvate (water removed)	Enolase	None
10	Phosphoenolpyruvate → pyruvate	Pyruvate kinase	1 ATP made

Steps 6 to 10 happen twice per glucose, once for each triose phosphate: 2 NADH and 4 ATP made, minus 2 ATP used in steps 1 and 3.

The link reaction: pyruvate to acetyl coenzyme A4 rows

Product	Per pyruvate	Per glucose	What happens to it
Acetyl CoA	1	2	Enters the Krebs cycle
CO₂	1	2	Diffuses out; the first CO₂ of aerobic respiration
NADH	1	2	Reoxidised by the respiratory chain
ATP	0	0	No ATP is made in this step

Watch out for (4)

Gross is not net
→ Glycolysis: the ten steps from glucose to pyruvate
Glycolysis releases no CO₂
→ Glycolysis: the ten steps from glucose to pyruvate
Isomerases rearrange; kinases phosphorylate
→ Glycolysis: the ten steps from glucose to pyruvate
Decarboxylation releases CO₂ but uses no oxygen
→ The link reaction: pyruvate to acetyl coenzyme A

Drill the past-year questions on this topic

Open the bank with this topic pre-filtered.

Open in the bank