Glycolysis is a pathway that occurs in the cytosol, the aqueous interior of a cell outside membrane-bounded organelles. It converts one six-carbon glucose molecule into two three-carbon pyruvate molecules while capturing part of the chemical free-energy change as ATP and NADH.

It does not require molecular oxygen as a reactant.

The pathway has two bookkeeping phases.

Energy-investment and cleavage phase

Early reactions use ATP to phosphorylate and rearrange glucose. Two ATP molecules are consumed per glucose before the six-carbon sugar is cleaved into two three-carbon phosphorylated molecules.

A simplified carbon view is

glucose, 6 C
   ↓ consume 2 ATP in preparatory reactions
fructose-1,6-bisphosphate, 6 C
   ↓ cleavage and rearrangement
2 glyceraldehyde-3-phosphate, 3 C each

Phosphorylation helps retain sugar intermediates in the cell and changes their reactivity so that later cleavage and energy capture become possible.

Payoff phase

Each glyceraldehyde-3-phosphate is oxidized. NAD$^+$ accepts reducing equivalents, producing NADH, while the pathway creates reactive phosphorylated intermediates that can form ATP by substrate-level phosphorylation.

Because there are two three-carbon molecules per original glucose, the payoff reactions occur twice.

Per glucose, the payoff phase produces

• $2$ NADH;
• $4$ ATP by substrate-level phosphorylation;
• $2$ pyruvate.

Subtracting the two ATP invested earlier gives

$$\boxed{\text{net ATP}=4-2=2}.$$

A common net bookkeeping equation is therefore

$$\mathrm{glucose+2NAD^+ +2ADP+2P_i \rightarrow2pyruvate+2NADH+2H^+ +2ATP+2H_2O}.$$

Glycolysis only partially oxidizes glucose

No carbon is released as $\mathrm{CO_2}$ during glycolysis. All six glucose carbons remain in the two pyruvate molecules.

The pathway has therefore captured only part of the available free-energy decrease:

some → net ATP
some → NADH reducing power
much → remains in pyruvate

Under respiratory conditions, pyruvate can be oxidized further and NADH can transfer electrons to an electron-transport chain. When respiratory electron transfer is unavailable, cells need another route to regenerate NAD$^+$ so glycolysis can continue.

Pathway regulation is concentrated at strongly driven steps

Several glycolytic reactions operate far from equilibrium under cellular conditions and are common control points. Rather than assuming one universal rate-limiting reaction, the useful principle is that pathway flux can be regulated at strategically strongly driven steps in response to substrate supply and cellular demand.

Glycolysis is therefore both a carbon-conversion pathway and an energy-capture pathway: glucose is split and partially oxidized to pyruvate while ATP and NADH are produced.