structural:summarization, structural:expansion
Pay-off phase

The second half of glycolysis is known as the pay-off phase, characterised by a net gain of the energy-rich molecules ATP and NADH. Since glucose leads to two triose sugars in the preparatory phase, each reaction in the pay-off phase occurs twice per glucose molecule. This yields 2 NADH molecules and 4 ATP molecules, leading to a net gain of 2 NADH molecules and 2 ATP molecules from the glycolytic pathway per glucose.

The aldehyde groups of the triose sugars are oxidised, and inorganic phosphate is added to them, forming 1,3-bisphosphoglycerate.

The hydrogen is used to reduce two molecules of NAD, a hydrogen carrier, to give NADH + H for each triose.

Hydrogen atom balance and charge balance are both maintained because the phosphate (P) group actually exists in the form of a hydrogen phosphate anion (HPO 2− 4), which dissociates to contribute the extra H ion and gives a net charge of -3 on both sides.

Here, arsenate (), an anion akin to inorganic phosphate may replace phosphate as a substrate to form 1-arseno-3-phosphoglycerate. This, however, is unstable and readily hydrolyzes to form 3-phosphoglycerate, the intermediate in the next step of the pathway. As a consequence of bypassing this step, the molecule of ATP generated from 1-3 bisphosphoglycerate in the next reaction will not be made, even though the reaction proceeds. As a result, arsenate is an uncoupler of glycolysis.

This step is the enzymatic transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP by phosphoglycerate kinase, forming ATP and 3-phosphoglycerate. At this step, glycolysis has reached the break-even point: 2 molecules of ATP were consumed, and 2 new molecules have now been synthesized. This step, one of the two substrate-level phosphorylation steps, requires ADP; thus, when the cell has plenty of ATP (and little ADP), this reaction does not occur. Because ATP decays relatively quickly when it is not metabolized, this is an important regulatory point in the glycolytic pathway.

ADP actually exists as ADPMg, and ATP as ATPMg, balancing the charges at −5 both sides.

Cofactors: Mg

Phosphoglycerate mutase isomerises 3-phosphoglycerate into 2-phosphoglycerate.

Enolase next converts 2-phosphoglycerate to phosphoenolpyruvate. This reaction is an elimination reaction involving an E1cB mechanism.

Cofactors: 2 Mg, one "conformational" ion to coordinate with the carboxylate group of the substrate, and one "catalytic" ion that participates in the dehydration.

A final substrate-level phosphorylation now forms a molecule of pyruvate and a molecule of ATP by means of the enzyme pyruvate kinase. This serves as an additional regulatory step, similar to the phosphoglycerate kinase step.

Cofactors: Mg