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Biochemistry · Metabolic pathway

Glycolysis !

Glycolysis splits one glucose into two pyruvates in the cytosol, leaving a net 2 ATP and 2 NADH; it runs with or without oxygen.

Updated 2026-10-31

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Balance: ATP +2NADH +2pyruvate +2

Key facts

  • It happens in the cytosol of every cell; the red blood cell, which has no mitochondria, relies on it alone.
  • Investment phase (steps 1–5): 2 ATP spent. Payoff phase (steps 6–10): 4 ATP and 2 NADH made. Net: 2 ATP, 2 NADH, 2 pyruvate.
  • Three steps are irreversible and regulated: hexokinase, phosphofructokinase-1 (the committed step) and pyruvate kinase.
  • Without oxygen, lactate dehydrogenase turns pyruvate into lactate and regenerates the NAD⁺ glycolysis needs to keep going.
  • ATP is made by substrate-level phosphorylation (steps 7 and 10), with no respiratory chain.
Also called
Embden–Meyerhof pathway

Regulation

  • PFK-1 is the main control: AMP and fructose 2,6-bisphosphate activate it; ATP, citrate and acidity inhibit it.
  • Insulin raises fructose 2,6-bisphosphate in the liver and speeds glycolysis; glucagon lowers it.
  • Hexokinase is slowed by its product, glucose 6-phosphate; liver glucokinase is not.
  • Fructose 1,6-bisphosphate activates pyruvate kinase ahead of time; ATP, alanine and (in the liver) glucagon slow it.

The steps, one by one

  1. Step 1 · Hexokinase (glucokinase in liver and pancreas) irreversible Glucose + ATP → Glucose 6-phosphate + ADPPhosphorylates glucose at carbon 6: charged, it can no longer leave the cell.If it fails: Glucokinase mutations: MODY 2 (mild, stable hyperglycemia); hexokinase deficiency: a rare hemolytic anemia. Type 2 diabetes mellitus →
  2. Step 2 · Phosphoglucose isomerase Glucose 6-phosphate ⇌ fructose 6-phosphateTurns an aldose into a ketose: the six-membered ring becomes a five-membered one and C1 is free for another phosphate.If it fails: Glucose-6-phosphate isomerase deficiency: the second enzyme cause of non-spherocytic hemolytic anemia.
  3. Step 3 · Phosphofructokinase-1 (PFK-1) irreversible fructose 6-phosphate + ATP → Fructose 1,6-bisphosphate + ADPThe committed, rate-limiting step: once made, fructose 1,6-bisphosphate can only go on through glycolysis.If it fails: Muscle PFK deficiency (Tarui disease): cramps, myoglobinuria and mild hemolysis. Tarui disease (muscle phosphofructokinase deficiency) →
  4. Step 4 · Aldolase Fructose 1,6-bisphosphate ⇌ dihydroxyacetone phosphate + glyceraldehyde 3-phosphateSplits the six-carbon sugar into two triose phosphates (a reverse aldol reaction).If it fails: Aldolase B (liver): its deficiency causes hereditary fructose intolerance; aldolase A: rare myopathy and hemolysis.
  5. Step 5 · Triose phosphate isomerase dihydroxyacetone phosphate ⇌ glyceraldehyde 3-phosphateTurns dihydroxyacetone phosphate into glyceraldehyde 3-phosphate: from here everything happens twice per glucose.If it fails: TPI deficiency: hemolytic anemia with progressive neurological disease.
  6. Step 6 · Glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde 3-phosphate + NAD⁺ + phosphate (Pi) ⇌ 1,3-bisphosphoglycerate + NADHOxidises the aldehyde and adds an inorganic phosphate: the only step that makes NADH.If it fails: Arsenic poisoning: arsenate uncouples this step (and arsenite blocks PDH). Arsenic poisoning →
  7. Step 7 · Phosphoglycerate kinase 1,3-bisphosphoglycerate + ADP ⇌ 3-phosphoglycerate + ATPFirst substrate-level phosphorylation: the high-energy phosphate goes to ADP.If it fails: In red cells, bisphosphoglycerate mutase diverts 1,3-BPG to 2,3-BPG (no ATP), which tunes hemoglobin. PGK1 deficiency: hemolysis, myopathy.
  8. Step 8 · Phosphoglycerate mutase 3-phosphoglycerate ⇌ 2-phosphoglycerateMoves the phosphate from C3 to C2, via a phosphorylated histidine and 2,3-BPG.If it fails: Muscle PGAM deficiency (glycogen storage disease type X): exercise intolerance and myoglobinuria.
  9. Step 9 · Enolase 2-phosphoglycerate ⇌ phosphoenolpyruvate + H₂ORemoves a water molecule and makes phosphoenolpyruvate, a very high-energy compound.If it fails: Neuron-specific enolase (NSE) is used as a marker of small-cell lung cancer and neuroblastoma.
  10. Step 10 · Pyruvate kinase irreversible phosphoenolpyruvate + ADP → Pyruvate + ATPSecond substrate-level phosphorylation; the enol becomes a ketone and the reaction cannot go back.If it fails: Pyruvate kinase deficiency: the most common enzyme cause of non-spherocytic hemolytic anemia. Pyruvate kinase deficiency →
  11. Without oxygen (LDH) · Lactate dehydrogenase (without oxygen) Pyruvate + NADH ⇌ Lactate + NAD⁺Reduces pyruvate to lactate and returns NAD⁺ to step 6: that is how glycolysis keeps going without oxygen.If it fails: When lactate is made faster than the liver can use it, lactic acidosis follows. Lactic acidosis →

Clinical notes

  • Defects of red-cell glycolytic enzymes cause non-spherocytic hemolytic anemia; the most common is pyruvate kinase deficiency.
  • Tumours burn a lot of glucose even with oxygen (Warburg effect); FDG-PET uses the fact that hexokinase traps labelled 2-deoxyglucose.
  • The fluoride in grey-top tubes inhibits enolase: the blood stops using glucose and the reading stays accurate.
  • Arsenate uncouples step 6: glycolysis goes on but leaves no net ATP.

Test yourself

Think of the answer, then tap the question to see it.

What is the net yield of glycolysis per glucose?

2 ATP, 2 NADH and 2 pyruvate: 2 ATP are spent (steps 1 and 3) and 4 are made (steps 7 and 10).

Which enzyme catalyses the committed step of glycolysis?

Phosphofructokinase-1; AMP and fructose 2,6-bisphosphate activate it, ATP and citrate inhibit it.

Why does the red blood cell need lactate dehydrogenase?

It has no mitochondria: only LDH regenerates the NAD⁺ that step 6 uses up.

What does arsenate do to glycolysis?

It replaces phosphate at GAPDH; the product falls apart and the ATP of step 7 is lost, leaving no net ATP.

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Educational information, not medical advice. For any symptom, see a health professional.

Sources: Glycolysis (Reactome, 2026); Textbook of Biochemistry for Medical Students, 6th ed. (Jaypee Brothers Medical Publishers (Vasudevan DM, Sreekumari S, Vaidyanathan K), 2011); Lactic acidosis: MedlinePlus Medical Encyclopedia (MedlinePlus, US National Library of Medicine, 2026).

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