Glycolysis: Pathway and Net Yield

In short: Glycolysis is a ten-reaction pathway that converts one glucose molecule into two pyruvate molecules. It uses two ATP in the first phase and produces four ATP and two NADH in the second phase, for a net yield of two ATP and two NADH per glucose.

What glycolysis does

The Embden–Meyerhof pathway, commonly called glycolysis, is a sequence of enzyme-catalysed reactions. It has an ATP-investment phase and an ATP-generating phase. The six-carbon glucose molecule is first rearranged and split into two three-carbon molecules; the remaining reactions then occur twice per glucose.

Glycolysis pathway diagram

ATP-investment phase

Two ATP molecules are used to prepare glucose for cleavage. The products of the first five steps include two molecules of glyceraldehyde-3-phosphate (G3P), which continue into the second phase.

Step	Main conversion	Enzyme	Energy change per glucose
1	Glucose → glucose-6-phosphate	Hexokinase (or glucokinase in some tissues)	1 ATP used
2	Glucose-6-phosphate → fructose-6-phosphate	Phosphoglucose isomerase	None
3	Fructose-6-phosphate → fructose-1,6-bisphosphate	Phosphofructokinase-1	1 ATP used
4	Fructose-1,6-bisphosphate → glyceraldehyde-3-phosphate + dihydroxyacetone phosphate	Aldolase	None
5	Dihydroxyacetone phosphate ⇌ glyceraldehyde-3-phosphate	Triose-phosphate isomerase	None; leaves 2 G3P molecules

ATP-generating phase

Each of the two G3P molecules passes through these five reactions. ATP is formed directly in steps 7 and 10; the oxidation in step 6 also reduces NAD⁺ to NADH.

Step	Main conversion	Enzyme	Products per glucose
6	G3P → 1,3-bisphosphoglycerate	Glyceraldehyde-3-phosphate dehydrogenase	2 NADH
7	1,3-bisphosphoglycerate → 3-phosphoglycerate	Phosphoglycerate kinase	2 ATP
8	3-phosphoglycerate → 2-phosphoglycerate	Phosphoglycerate mutase	None
9	2-phosphoglycerate → phosphoenolpyruvate (PEP)	Enolase	2 water molecules released
10	PEP → pyruvate	Pyruvate kinase	2 ATP

Net yield per glucose

Four ATP are formed in the second phase and two ATP were used in the first. The net yield is therefore 2 ATP, 2 NADH and 2 pyruvate molecules per glucose. The two three-carbon molecules explain why each reaction from step 6 onward occurs twice.

Pharmaceutical record-retention note

When records are created as part of GMP activities, batch documentation in EU GMP scope must be retained for at least one year after the batch expiry or five years after Qualified Person certification, whichever is longer. Under US cGMP, batch-associated production, control or distribution records required by 21 CFR Part 211 must be retained for at least one year after batch expiry, subject to the regulation’s specific exceptions. EU GMP Part I, Chapter 4, §4.11 (January 2011); 21 CFR §211.180(a), current eCFR (accessed 16 October 2026)

For connected pathways, see gluconeogenesis, the TCA cycle and the electron transport chain.

Frequently asked questions

Why are steps 6 to 10 counted twice?

One glucose molecule is split into two three-carbon molecules. Both are converted through the same final five reactions, so each reaction produces two copies of its per-molecule product.

Why is the net ATP yield two instead of four?

The pathway forms four ATP during the second phase, but uses two ATP during the first phase. Subtracting the ATP investment gives a net gain of two ATP per glucose.

Does glycolysis produce lactate?

Glycolysis ends with pyruvate. In some cells and conditions, a subsequent reaction converts pyruvate to lactate; that reaction is separate from the ten glycolytic steps described here.

References

European Commission. EudraLex Volume 4, EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, Part I, Chapter 4: Documentation, §4.11, revision 1, January 2011. Official Chapter 4 PDF.
United States. Code of Federal Regulations, Title 21, Part 211, §211.180(a), General requirements, current eCFR, accessed 16 October 2026. Official eCFR text.