Biochemistry · Metabolism · Cheat sheet
Glycolysis steps cheat sheet: all 10 enzymes, ATP, and regulation
Glycolysis splits one glucose (6 carbons) into two pyruvate (3 carbons each) in the cytoplasm, in 10 steps. It spends 2 ATP, makes 4, and nets 2 ATP, 2 NADH and 2 pyruvate per glucose. Everything you need for an exam is on this page, and the practice at the bottom checks it.
- 10 steps
- 2 ATP spent (steps 1, 3)
- 4 ATP made (steps 7, 10)
- 2 NADH (step 6)
- 3 one-way steps (1, 3, 10)
Glucose + 2 NAD⁺ + 2 ADP + 2 Pᵢ → 2 pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 H₂O
Jump to: the 10-step table, regulation, worked examples, practice.
All 10 steps on one table
Read it row by row once. Then use the buttons to hide a column and test yourself: each hidden cell gets a Show button, so you can check one answer at a time.
Hide a column to quiz yourself:
| Step | Reaction | Enzyme | Reaction type | ATP / NADH | One-way? |
|---|---|---|---|---|---|
| 1 | Glucose (6C) → glucose-6-phosphate | Hexokinase (glucokinase in liver and pancreas) | Phosphorylation. A phosphate from ATP goes onto the sugar. | Uses 1 ATP | One-way, regulated |
| 2 | Glucose-6-phosphate → fructose-6-phosphate | Phosphoglucose isomerase | Isomerization. An aldose becomes a ketose. | None | Reversible |
| 3 | Fructose-6-phosphate → fructose-1,6-bisphosphate | Phosphofructokinase-1 (PFK-1) | Phosphorylation. The committed step. | Uses 1 ATP | One-way, main control point |
| 4 | Fructose-1,6-bisphosphate (6C) → DHAP (3C) + glyceraldehyde-3-phosphate (3C) | Aldolase | Cleavage. One 6-carbon sugar splits into two 3-carbon pieces. | None | Reversible |
| 5 | DHAP → glyceraldehyde-3-phosphate (G3P) | Triose phosphate isomerase | Isomerization. Now both halves are G3P, so both can finish. | None | Reversible |
| 6 | G3P + NAD⁺ + Pᵢ → 1,3-bisphosphoglycerate + NADH | Glyceraldehyde-3-phosphate dehydrogenase | Oxidation. The new phosphate comes from Pᵢ, not ATP. | Makes 1 NADH (2 per glucose) | Reversible |
| 7 | 1,3-Bisphosphoglycerate + ADP → 3-phosphoglycerate + ATP | Phosphoglycerate kinase | Substrate-level phosphorylation. First ATP made. | Makes 1 ATP (2 per glucose) | Reversible |
| 8 | 3-Phosphoglycerate → 2-phosphoglycerate | Phosphoglycerate mutase | Phosphate shift from carbon 3 to carbon 2. | None | Reversible |
| 9 | 2-Phosphoglycerate → phosphoenolpyruvate (PEP) + H₂O | Enolase | Dehydration. Water leaves. | None | Reversible |
| 10 | PEP + ADP → pyruvate (3C) + ATP | Pyruvate kinase | Substrate-level phosphorylation. Second ATP made. | Makes 1 ATP (2 per glucose) | One-way, regulated |
The three shaded rows marked "One-way" are the control points. In the cell they have large negative free-energy changes, so they are where the pathway is controlled, and gluconeogenesis needs different enzymes to get around them.
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The two halves in one line each
Investment: steps 1 to 5
Spend 2 ATP to put a phosphate on each end of the sugar, then split it into two 3-carbon pieces and turn both into G3P.
Payoff: steps 6 to 10, twice
Each G3P makes 1 NADH and 2 ATP on its way to pyruvate. Two G3P make 2 NADH and 4 ATP. Subtract the 2 you spent: net 2 ATP.
Regulation: the three one-way steps
The pattern is about energy. Signs that the cell has plenty of energy (ATP, citrate) slow glycolysis down. Signs that it is running low (AMP) speed it up. PFK-1 is the main control point.
| Enzyme | Speeds it up | Slows it down | Hormones and the liver | Gluconeogenesis goes around it with |
|---|---|---|---|---|
| Hexokinase (step 1) | No major activator; it runs faster when more glucose is available | Glucose-6-phosphate, its own product | Liver and pancreas use glucokinase, which has a higher Km and is not inhibited by glucose-6-phosphate. | Glucose-6-phosphatase |
| PFK-1 (step 3) | AMP (and ADP), fructose-2,6-bisphosphate | ATP, citrate, acidic pH | Insulin raises fructose-2,6-bisphosphate. In the liver, glucagon lowers it: protein kinase A phosphorylates PFK-2/FBPase-2, which switches off the kinase half and switches on the phosphatase half. | Fructose-1,6-bisphosphatase |
| Pyruvate kinase (step 10) | In liver, fructose-1,6-bisphosphate (feed-forward) | ATP, alanine | In the liver, glucagon leads protein kinase A to phosphorylate pyruvate kinase, which turns it off. | Pyruvate carboxylase, then PEP carboxykinase (PEPCK) |
ATP plays two parts at PFK-1. It is a substrate, but at high levels it also binds a separate regulatory site that turns the enzyme down.
Memory aids
The 11 molecules, in order: "Goodness Gracious, Father Franklin Did Go By Picking Pumpkins (to) Prepare Pies." That is glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, DHAP, G3P, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, PEP, pyruvate.
The enzymes, by job: once you know the molecules, most enzyme names follow. Kinases move a phosphate between ATP (or ADP) and a substrate (steps 1, 3, 7, 10). Isomerases rearrange (steps 2, 5). Aldolase splits (step 4). The dehydrogenase oxidizes (step 6). The mutase moves a phosphate within one molecule (step 8). Enolase pulls out water to make the "enol" in phosphoenolpyruvate (step 9).
Five exam traps
- The kinases at steps 7 and 10 make ATP. Phosphoglycerate kinase and pyruvate kinase are named for the reverse reaction, but in glycolysis they move a phosphate onto ADP.
- Steps 6 to 10 count twice. If you get 1 NADH or 2 ATP made, you forgot that each glucose gives two G3P.
- Gross is not net. 4 ATP made, 2 spent, 2 net.
- Step 6 adds a phosphate without ATP. It uses inorganic phosphate (Pᵢ). Only steps 1 and 3 spend ATP.
- Glucokinase is not just hexokinase in another tissue. It has a higher Km and is not inhibited by glucose-6-phosphate, so liver cells can keep phosphorylating glucose after a meal.
Worked examples
Example 1: Glucose from glycogen nets 3 ATP
Muscle breaks down glycogen during exercise. Each glucose unit comes off as glucose-1-phosphate, which is converted to glucose-6-phosphate. How much net ATP does glycolysis make from one of these glucose units?
- Find where it enters. It is already glucose-6-phosphate, so it starts at step 2. Hexokinase is skipped.
- ATP spent: only step 3 (PFK-1), so 1 ATP.
- ATP made: the payoff phase does not change. 2 at step 7 + 2 at step 10 = 4 ATP.
- Net: 4 − 1 = 3 ATP.
Answer: 3 ATP (and still 2 NADH) per glucose unit from glycogen, compared with 2 ATP for free glucose.
Example 2: Keep the books balanced during a sprint
A sprinter's leg muscle is short on oxygen. Track NAD⁺ and NADH for one glucose. Why does the muscle make lactate?
- Step 6 runs twice and turns 2 NAD⁺ into 2 NADH.
- The cell holds only a small pool of NAD⁺. Without oxygen, the electron transport chain cannot reoxidize NADH, so that pool would run dry and step 6 would stop.
- Lactate dehydrogenase reduces each pyruvate to lactate, and in doing so oxidizes 1 NADH back to NAD⁺. Two pyruvate fix 2 NADH.
- Tally: 2 ATP net, 0 NADH left over, 2 lactate.
Answer: lactate is made to give NAD⁺ back to step 6. Fermentation makes no extra ATP; it keeps the 2 ATP from glycolysis coming.
Example 3: Predict the liver after a meal and after a fast
Does glycolysis in the liver speed up or slow down (a) after a carbohydrate meal, when insulin is high, and (b) after an overnight fast, when glucagon is high?
- (a) Insulin activates a protein phosphatase that dephosphorylates PFK-2/FBPase-2. The kinase half turns on and makes more fructose-2,6-bisphosphate, which activates PFK-1. Glycolysis speeds up.
- (b) Glucagon raises cAMP, which activates protein kinase A. PKA phosphorylates PFK-2/FBPase-2, so the phosphatase half breaks down fructose-2,6-bisphosphate and PFK-1 loses its activator.
- PKA also phosphorylates liver pyruvate kinase, which turns it off, so PEP is not burned to pyruvate.
Answer: (a) speeds up; (b) slows down at steps 3 and 10, so the fasting liver can make glucose by gluconeogenesis instead of burning it.
Practice
Three short sets. Try them without scrolling up. Every item gives instant feedback and has a full solution.
Set 1: Put the 11 molecules in order
These are scrambled. Sort them from glucose at the top to pyruvate at the bottom. Use the Up and Down buttons, then press Check order.
- Phosphoenolpyruvate (PEP)
- DHAP
- Glucose-6-phosphate
- Pyruvate
- Fructose-1,6-bisphosphate
- 2-Phosphoglycerate
- Glucose
- 1,3-Bisphosphoglycerate
- Fructose-6-phosphate
- 3-Phosphoglycerate
- Glyceraldehyde-3-phosphate
Show the correct order
Glucose, Glucose-6-phosphate, Fructose-6-phosphate, Fructose-1,6-bisphosphate, DHAP, Glyceraldehyde-3-phosphate, 1,3-Bisphosphoglycerate, 3-Phosphoglycerate, 2-Phosphoglycerate, Phosphoenolpyruvate (PEP), Pyruvate.
Set 2: Name that step
Pick the step number (1 to 10) that matches each clue, then press Check.
Set 3: Exam-style scenarios
Frequently asked questions
How many steps are in glycolysis, and how much ATP does it make?
Ten steps. Glycolysis makes 4 ATP but spends 2, so the net is 2 ATP per glucose, plus 2 NADH and 2 pyruvate. The 2 ATP are spent at steps 1 and 3, and the 4 are made at steps 7 and 10, each of which runs twice.
Where does glycolysis happen, and does it need oxygen?
In the cytoplasm, in both prokaryotic and eukaryotic cells. No step uses oxygen. But step 6 needs NAD⁺, so the cell has to keep turning NADH back into NAD⁺, by aerobic respiration when oxygen is present, or by fermentation or anaerobic respiration when it is not.
What happens to pyruvate after glycolysis?
In human cells with enough oxygen, pyruvate moves into the mitochondria, where pyruvate dehydrogenase turns it into acetyl-CoA for the citric acid cycle. Without enough oxygen, human muscle uses lactate dehydrogenase to turn pyruvate into lactate, and yeast makes ethanol and CO₂. Both fermentation routes exist to regenerate NAD⁺ so glycolysis can keep going.
Why is step 3 the committed step and not step 1?
Glucose-6-phosphate from step 1 has other places to go, such as glycogen or the pentose phosphate pathway. Fructose-1,6-bisphosphate from step 3 has no other major use, so once PFK-1 acts, the sugar is headed down glycolysis. That is why PFK-1 is the main control point.
What is an easy way to remember the glycolysis steps?
Learn the 11 molecules with a sentence like "Goodness Gracious, Father Franklin Did Go By Picking Pumpkins to Prepare Pies," then hang the enzymes on them by job: kinases at steps 1, 3, 7 and 10, isomerases at 2 and 5, aldolase splits at 4, the one dehydrogenase at 6, the mutase at 8 and enolase at 9.
Sources
- OpenStax Biology 2e, 7.2 Glycolysis (steps, location, yield, NAD⁺)
- OpenStax Biology 2e, 7.5 Metabolism Without Oxygen (lactate and ethanol fermentation)
- OpenStax Biology 2e, 7.6 Connections of Carbohydrate, Protein, and Lipid Metabolic Pathways (glycogen to glucose-6-phosphate)
- OpenStax Biology 2e, 7.7 Regulation of Cellular Respiration (control at steps 1, 3 and 10)
- OpenStax Anatomy and Physiology 2e, 24.2 Carbohydrate Metabolism (glucokinase, fate of pyruvate, gluconeogenesis bypass enzymes)
- LibreTexts, Fundamentals of Biochemistry (Jakubowski and Flatt), 15.4 Regulation of Glycolysis (glucokinase, PFK-2/FBPase-2, insulin and glucagon, pyruvate kinase)
- Jitrapakdee et al., Structure, Mechanism and Regulation of Pyruvate Carboxylase (first gluconeogenesis bypass step)
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