Glycolysis and the Transition Reaction

Glycolysis is the ten-step pathway in the cytoplasm that splits one glucose molecule (6 carbons) into two pyruvate molecules (3 carbons each), with a net gain of 2 ATP and 2 NADH, and the transition (link) reaction then converts pyruvate into acetyl-CoA in the mitochondrial matrix. In brief: glycolysis needs no oxygen and happens in nearly all living cells. It has an investment phase, which uses 2 ATP, and a payoff phase, which produces 4 ATP and 2 NADH. The link reaction removes one carbon as CO₂ and produces one NADH per pyruvate.

These are the first stages of aerobic respiration, and an introduction to oxidation and reduction in metabolism. This guide reviews ATP and redox reactions, follows the ten steps of glycolysis in three groups, explains the link reaction, compares the stages, works through an ATP and NADH accounting example and corrects common mistakes.

What are oxidation and reduction in metabolism?

Oxidation is the loss of electrons (or of hydrogen), and reduction is the gain of electrons (or of hydrogen). They occur together in redox reactions. In respiration, glucose is oxidized, and coenzymes are reduced. The coenzyme NAD⁺ accepts two electrons and one hydrogen ion, with another hydrogen ion released into solution, to become NADH: NAD⁺ + 2e⁻ + 2H⁺ → NADH + H⁺. NADH then carries the electrons to the electron transport chain, where their energy is used to make ATP. Because the cell has only a small amount of NAD⁺, it must be regenerated, either by the electron transport chain (with oxygen) or by fermentation (without).

What happens in glycolysis?

The word glycolysis means “splitting of sugar”. It occurs in the cytosol and has ten steps, each catalyzed by a specific enzyme. The net equation is:

glucose + 2NAD⁺ + 2ADP + 2Pᵢ → 2 pyruvate + 2NADH + 2H⁺ + 2ATP + 2H₂O

Energy-investment phase (steps 1 to 5)

  1. Hexokinase transfers a phosphate from ATP to glucose to make glucose-6-phosphate. (1 ATP used.) The phosphate traps glucose in the cell.
  2. Glucose-6-phosphate is rearranged to fructose-6-phosphate.
  3. Phosphofructokinase transfers a second phosphate from ATP to form fructose-1,6-bisphosphate. (1 ATP used.) This is the main control point.
  4. The six-carbon sugar is split into two three-carbon sugars: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate, which is converted into G3P.
  5. The result is two molecules of G3P, which go through the next steps. Every step in the payoff phase happens twice per glucose.

Energy-payoff phase (steps 6 to 10)

  1. Each G3P is oxidized, and NAD⁺ is reduced to NADH. A phosphate group is added, giving 1,3-bisphosphoglycerate.
  2. The phosphate is transferred to ADP, making ATP, by substrate-level phosphorylation, and 3-phosphoglycerate is formed.
  3. Rearrangement to 2-phosphoglycerate.
  4. Water is removed to give phosphoenolpyruvate (PEP).
  5. The last phosphate is transferred from PEP to ADP, making ATP and pyruvate.

Glycolysis in three groups of steps

Energy accounting in glycolysis (per glucose)
Phase ATP used ATP made NADH made Main product
Investment (steps 1 to 5)2002 G3P
Payoff (steps 6 to 10)0422 pyruvate
Net+2 ATP22 pyruvate
Net ATP after each step of glycolysis per glucose 1 2 3 4 5 6 7 8 9 10 Glycolysis step Net ATP (cumulative) −3 −2 −1 0 +1 +2 +3 Investment: 2 ATP used Payoff: 4 ATP made net +2
Running total of ATP per glucose after each of the ten steps. Steps 1 and 3 each use one ATP, so the total falls to minus 2. Steps 7 and 10 each make two ATP (one per triose phosphate), taking the total to zero and then to a net gain of +2, as in the table.

How is glycolysis regulated?

The pathway is controlled by feedback. Phosphofructokinase is inhibited by high levels of ATP and citrate (signals that the cell has plenty of energy), and stimulated by AMP and ADP (signals of low energy). Hexokinase is inhibited by its product, glucose-6-phosphate. This ensures that glucose is only used as quickly as the cell needs the energy.

If oxygen is available, pyruvate is transported from the cytosol into the mitochondrial matrix. There, the pyruvate dehydrogenase complex converts it in three coupled changes:

  1. Decarboxylation: one carbon is removed as CO₂ (leaving a 2-carbon acetyl group).
  2. Oxidation: the acetyl group is oxidized, and NAD⁺ is reduced to NADH.
  3. The acetyl group attaches to coenzyme A to form acetyl-CoA.

For one pyruvate: pyruvate + NAD⁺ + CoA → acetyl-CoA + NADH + CO₂ + H⁺. Because one glucose gives two pyruvates, the link reaction yields 2 acetyl-CoA, 2 NADH and 2 CO₂ per glucose. No ATP is made directly. The acetyl-CoA then enters the Krebs cycle, which is described in the next article. Without oxygen, pyruvate is converted to lactate or ethanol (fermentation) in the cytosol instead.

C₂ CO₂ C₂ CO₂

Worked example: tracking carbon, ATP and NADH

Account for one molecule of glucose (C₆H₁₂O₆) through glycolysis and the link reaction.

  1. Carbons: glucose has 6 carbons. Glycolysis produces 2 pyruvate (3 carbons each = 6 carbons). The link reaction removes 1 carbon from each pyruvate, giving 2 CO₂ (2 carbons) and 2 acetyl groups (2 carbons each = 4 carbons). Total: 2 + 4 = 6 carbons. All carbons are accounted for.
  2. ATP in glycolysis: used 2, made 4, net +2.
  3. NADH: 2 from glycolysis and 2 from the link reaction = 4 NADH.
  4. Energy in NADH: each NADH can drive the production of about 2.5 ATP via the electron transport chain (current estimates). So 4 NADH could yield about 4 × 2.5 = 10 ATP. Add the 2 ATP from glycolysis: about 12 ATP so far, from the first two stages.

This is less than half of the full yield from respiration (about 30 to 32 ATP), as the Krebs cycle and the electron transport chain give the rest. The NADH produced in the cytosol has to be shuttled into the mitochondrion, which costs some energy, so the 2.5 ATP per NADH figure for the cytosolic NADH is approximate. Older textbooks use 3 ATP per NADH.

How was glycolysis discovered?

Louis Pasteur showed that fermentation requires living yeast, and Eduard Buchner showed in 1897 that extracts from yeast can ferment sugar. Gustav Embden, Otto Meyerhof and Jakub Parnas worked out the pathway in the 1920s and 1930s, so it is also known as the Embden-Meyerhof-Parnas pathway. Meyerhof shared the 1922 Nobel Prize in Physiology or Medicine with Archibald Hill. Glycolysis is thought to be one of the oldest metabolic pathways, since it exists in nearly all organisms and does not need oxygen, which was scarce when life began.

Which mistakes are common?

Mistake 1: Saying that glycolysis makes 4 ATP net

It makes 4 but uses 2, so the net gain is 2.

Mistake 2: Putting glycolysis in the mitochondrion

It takes place in the cytosol. The link reaction is in the mitochondrial matrix.

Mistake 3: Forgetting that glucose gives two of everything after splitting

From step 4 onward, each reaction occurs twice for each glucose.

Mistake 4: Saying that the link reaction makes ATP

It makes NADH and CO₂ and acetyl-CoA, but no ATP directly.