Glycolysis: Which Steps Produce ATP and NADH

Which Steps of Glycolysis Make or Use ATP and NADH?

By StudyStore · Published 2026-11-09 · 1 min read · 298 words

Glycolysis has only four ATP or NADH events. Two steps spend ATP, one step makes NADH, and two steps make ATP, and the last three happen twice per glucose because the sugar splits into two three-carbon pieces.

The four steps to memorize

Step	Enzyme	Event
1. Glucose to glucose-6-phosphate	Hexokinase (glucokinase in liver)	Uses 1 ATP
3. Fructose-6-phosphate to fructose-1,6-bisphosphate	PFK-1	Uses 1 ATP
6. G3P to 1,3-bisphosphoglycerate	GAPDH	Makes 1 NADH (x2)
7. 1,3-BPG to 3-phosphoglycerate	Phosphoglycerate kinase	Makes 1 ATP (x2)
10. Phosphoenolpyruvate to pyruvate	Pyruvate kinase	Makes 1 ATP (x2)

What substrate-level phosphorylation means

The ATP made at steps 7 and 10 comes from transferring a phosphate directly from a high-energy substrate to ADP. No electron transport chain is involved. Oxidative phosphorylation is different: it uses the proton gradient made by the NADH and FADH₂ that the electron transport chain reoxidizes.

Net yield per glucose

Spent: 2 ATP. Made: 4 ATP. Net: 2 ATP and 2 NADH, plus 2 pyruvate.

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Common traps

Counting 4 ATP as the net. Subtract the 2 spent in the investment phase.
Forgetting to double the later steps. One glucose gives two G3P.
Saying NADH is made at the ATP steps. NADH forms at GAPDH only.

One-minute recap

ATP used: hexokinase and PFK-1.
NADH made: GAPDH. ATP made: PGK and pyruvate kinase.
Net per glucose: 2 ATP, 2 NADH, 2 pyruvate.

Related: see the net yield of glycolysis.

Check yourself: 5-question mini-quiz

Try each question first, then click it to reveal the answer.

1. Which two glycolysis enzymes use ATP?

Hexokinase and PFK-1.

2. Which enzyme produces NADH in glycolysis?

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

3. Which two enzymes make ATP by substrate-level phosphorylation?

Phosphoglycerate kinase and pyruvate kinase.

4. What is the net ATP per glucose?

2 ATP (4 made minus 2 spent).

5. Why are the later steps counted twice?

One glucose splits into two three-carbon molecules of G3P.

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Written by StudyStore from standard scientific facts. Study aid only. Always check values against the reference your exam provides.