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Biochemistry · Metabolism

Glycolysis quiz: the 10 steps, enzymes, and yield, with answers

Glycolysis splits one glucose into two pyruvate in 10 steps and nets 2 ATP and 2 NADH. The big idea: spend 2 ATP up front, split the sugar in half, then collect twice from each half. This quiz covers the order, the enzymes, the payoff, and how PFK-1 is controlled, the way college biochemistry exams ask it.

Already know the basics? Jump to the 8 practice questions or the worked examples.

The 10 steps in order

Glycolysis turns one glucose (6 carbons) into two pyruvate (3 carbons each) in the cytoplasm. It runs with or without oxygen. Here is every step, with what goes in, what comes out, and where ATP and NADH show up.

The 10 steps of glycolysis. Steps 6 to 10 run twice per glucose.
StepSubstrate → productEnzymeATP / NADH
1 (regulated)Glucose → Glucose-6-phosphateHexokinaseUses 1 ATP
2Glucose-6-phosphate → Fructose-6-phosphatePhosphoglucose isomeraseNone
3 (regulated)Fructose-6-phosphate → Fructose-1,6-bisphosphatePhosphofructokinase-1 (PFK-1)Uses 1 ATP
4Fructose-1,6-bisphosphate → DHAP + glyceraldehyde-3-phosphate (G3P)AldolaseNone
5DHAP → G3PTriose phosphate isomeraseNone
6G3P (+ NAD⁺ + Pᵢ) → 1,3-BisphosphoglycerateGlyceraldehyde-3-phosphate dehydrogenaseMakes 1 NADH (2 per glucose)
71,3-Bisphosphoglycerate → 3-PhosphoglyceratePhosphoglycerate kinaseMakes 1 ATP (2 per glucose)
83-Phosphoglycerate → 2-PhosphoglyceratePhosphoglycerate mutaseNone
92-Phosphoglycerate → Phosphoenolpyruvate (PEP) + H₂OEnolaseNone
10 (regulated)PEP → PyruvatePyruvate kinaseMakes 1 ATP (2 per glucose)

A quick memory aid for the enzyme order is the first letters: Hexokinase, Phosphoglucose isomerase, PFK-1, Aldolase, Triose phosphate isomerase, G3P dehydrogenase, Phosphoglycerate kinase, Phosphoglycerate mutase, Enolase, Pyruvate kinase (H-P-P-A-T-G-P-P-E-P). Make up a sentence that sticks for you.

Investment phase vs payoff phase

Steps 1 to 5 are the investment phase. The cell spends 2 ATP: one at hexokinase and one at PFK-1. Aldolase then splits the 6-carbon sugar into two 3-carbon pieces, DHAP and G3P. Triose phosphate isomerase converts DHAP into G3P, so you end up with two G3P.

Steps 6 to 10 are the payoff phase, and each step happens twice per glucose because there are two G3P.

  1. G3P dehydrogenase oxidizes G3P and makes 1 NADH each time: 2 NADH per glucose.
  2. Phosphoglycerate kinase makes 1 ATP each time: 2 ATP.
  3. Pyruvate kinase makes 1 ATP each time: 2 ATP.

Both ATP-making steps are substrate-level phosphorylation: an enzyme moves a phosphate group straight from a high-energy substrate onto ADP.

Net per glucose: 4 ATP made − 2 ATP used = 2 ATP, plus 2 NADH and 2 pyruvate

NADH has to be turned back into NAD⁺ for step 6 to keep going. With oxygen, the electron transport chain does that. Without oxygen, fermentation does it.

The three regulated steps and PFK-1 control

Three steps have large negative free-energy changes and are effectively one-way inside the cell: hexokinase (step 1), PFK-1 (step 3), and pyruvate kinase (step 10). Those are the control points. Gluconeogenesis uses different enzymes to get around these three steps.

How the three regulated enzymes are controlled
EnzymeInhibited byActivated by
HexokinaseGlucose-6-phosphate (its own product)No major allosteric activator; its rate follows glucose supply
PFK-1ATP, citrateAMP, fructose-2,6-bisphosphate
Pyruvate kinaseATP, alanineFructose-1,6-bisphosphate (feedforward)

PFK-1 is the committed step and the main control point. The logic is about energy. Lots of ATP means the cell has enough energy, so PFK-1 slows down. ATP is a substrate here too, but at high levels it also binds a separate regulatory site that turns the enzyme down. Citrate, from the citric acid cycle, also signals plenty and inhibits PFK-1. AMP builds up when energy is low, so it activates PFK-1. Fructose-2,6-bisphosphate, made by a separate enzyme (PFK-2), is a strong activator in eukaryotes.

Worked examples

Example 1: Count the ATP and NADH yourself

Without looking at a summary, find the net ATP and NADH from one glucose.

  1. Investment: hexokinase uses 1 ATP and PFK-1 uses 1 ATP. Total used: 2 ATP.
  2. Aldolase plus triose phosphate isomerase give 2 G3P, so steps 6 to 10 each run twice.
  3. Step 6: 1 NADH × 2 = 2 NADH.
  4. Step 7: 1 ATP × 2 = 2 ATP. Step 10: 1 ATP × 2 = 2 ATP. Total made: 4 ATP.
  5. Net ATP: 4 − 2 = 2.

Answer: net 2 ATP and 2 NADH (and 2 pyruvate) per glucose.

Example 2: Predict PFK-1 activity

A muscle cell starts sprinting. ATP drops and AMP rises. What happens to PFK-1 and to glycolysis?

  1. Less ATP means less inhibition at PFK-1's regulatory site.
  2. More AMP means more activation. AMP is the low-energy signal.
  3. Both changes push PFK-1 to work faster, so more fructose-1,6-bisphosphate is made.
  4. Fructose-1,6-bisphosphate also activates pyruvate kinase downstream, so the whole pathway speeds up.

Answer: PFK-1 speeds up and glycolysis flux increases, producing ATP faster.

Example 3: Name the enzyme from the structures

An enzyme moves a phosphate from carbon 3 to carbon 2 of a three-carbon sugar acid. Which step is it, and what comes next?

  1. Moving a group within the same molecule is a mutase reaction.
  2. 3-Phosphoglycerate → 2-phosphoglycerate is step 8, phosphoglycerate mutase.
  3. Next, enolase removes water from 2-phosphoglycerate to make phosphoenolpyruvate (PEP), step 9.
  4. Then pyruvate kinase transfers PEP's phosphate to ADP, step 10.

Answer: step 8, phosphoglycerate mutase; enolase (step 9) comes next.

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Put the glycolysis steps in order

Choose the enzyme for each step, from glucose (step 1) to pyruvate (step 10). Then press Check order. The table above has the answers, so try it before you scroll up.

Practice questions

Pick an answer, then check it. A wrong answer tells you what went wrong, so you can try again. Every question also has a full solution.

Question 1Which enzyme catalyzes the committed step of glycolysis?

Show solution

PFK-1 makes fructose-1,6-bisphosphate, which has no other major fate, so this is the committed step. It is the most tightly regulated enzyme in the pathway.

Question 2Which enzyme produces NADH in glycolysis?

Show solution

Step 6, G3P dehydrogenase, oxidizes G3P and adds inorganic phosphate to make 1,3-bisphosphoglycerate. NAD⁺ is reduced to NADH. It happens twice per glucose, for 2 NADH.

Question 3What is the net yield of glycolysis per molecule of glucose?

Show solution

Used: 2 ATP (steps 1 and 3). Made: 2 ATP at step 7 and 2 ATP at step 10, total 4. Net 2 ATP. Step 6 makes 2 NADH, and the pathway ends with 2 pyruvate.

Question 4Which molecule activates PFK-1?

Show solution

PFK-1 is activated by AMP and fructose-2,6-bisphosphate and inhibited by ATP and citrate. The activators signal low energy or plenty of glucose; the inhibitors signal plenty of energy.

Question 5Which two enzymes make ATP by substrate-level phosphorylation in glycolysis?

Show solution

Step 7 (phosphoglycerate kinase) and step 10 (pyruvate kinase) each make 1 ATP per three-carbon molecule, so 2 ATP each per glucose. Both transfer a phosphate group directly from a substrate to ADP, which is what substrate-level phosphorylation means.

Question 6Which enzyme is not one of the three regulated, effectively irreversible steps of glycolysis?

Show solution

The three regulated, effectively one-way steps are 1 (hexokinase), 3 (PFK-1), and 10 (pyruvate kinase). Phosphoglucose isomerase (step 2) is freely reversible.

Question 7Why does each payoff-phase step (steps 6 to 10) happen twice for every glucose?

Show solution

Aldolase cleaves fructose-1,6-bisphosphate into DHAP and G3P. Triose phosphate isomerase converts DHAP into G3P. Now there are two G3P per glucose, so steps 6 to 10 each run twice.

Question 8Which enzyme converts 2-phosphoglycerate into phosphoenolpyruvate?

Show solution

Step 9: enolase dehydrates 2-phosphoglycerate (removes H₂O) to form phosphoenolpyruvate, a high-energy phosphate compound. Pyruvate kinase then uses it in step 10.

Answer key

  1. B, PFK-1: makes fructose-1,6-bisphosphate, the committed step.
  2. D, G3P dehydrogenase: the one redox step.
  3. B, 2 ATP, 2 NADH, 2 pyruvate: 4 made − 2 used.
  4. C, fructose-2,6-bisphosphate: activator; ATP and citrate inhibit.
  5. B, phosphoglycerate kinase and pyruvate kinase: steps 7 and 10.
  6. D, phosphoglucose isomerase: reversible, not regulated.
  7. A: the aldolase split plus triose phosphate isomerase give two G3P.
  8. C, enolase: dehydration, step 9.

Frequently asked questions

Is glycolysis on the MCAT?

Yes. The AAMC content outline lists glycolysis under bioenergetics and metabolism. Expect the regulated enzymes, net yield, where NADH comes from, and how the pathway responds to energy signals. Knowing all 10 intermediates helps, but the regulation and the energy accounting come up most.

Do I need to memorize all 10 glycolysis enzymes?

Most college biochemistry courses expect the enzymes in order, and they are much easier to remember when you pair each with what it does: kinases move phosphates, isomerases rearrange, the dehydrogenase oxidizes, and the mutase shifts a phosphate within the molecule.

Which steps of glycolysis are irreversible?

Steps 1, 3, and 10, catalyzed by hexokinase, PFK-1, and pyruvate kinase. They have large negative free-energy changes in the cell, so they act as one-way control points, and gluconeogenesis uses different enzymes to get around them.

Does glycolysis need oxygen?

No. Glycolysis itself does not use oxygen. But step 6 needs NAD⁺, so the cell has to regenerate it, through the electron transport chain when oxygen is present or through fermentation when it is not.

Sources

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