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Glyceraldehyde phosphate dehydrogenase oxidizes the 3-carbon G3P molecules, extracting high-energy electrons that are captured by NAD+ to produce NADH. Simultaneously, a phosphate group is added to form 1,3-bisphosphoglycerate. This oxidation step is critical because the continuation depends on NAD+ availability; if NAD+ is unavailable, the pay-off phase slows or stops.

The pay-off phase produces four ATP molecules total. Phosphoglycerate kinase generates two ATP when 1,3-bisphosphoglycerate is dephosphorylated to 3-phosphoglycerate. Pyruvate kinase generates two more ATP when phosphoenolpyruvate is converted to pyruvate. Since the preparatory phase uses two ATP, the net yield of glycolysis is two ATP molecules.

Enolase catalyzes a dehydration reaction that removes water from 2-phosphoglycerate, creating a double bond in the remaining phosphate group. This increases the potential energy of the phosphate bond, producing phosphoenolpyruvate (PEP). The enhanced energy in PEP makes it available for the final ATP-generating step catalyzed by pyruvate kinase.

NAD+ is required to accept high-energy electrons during the oxidation of G3P in step 6. If NAD+ becomes depleted and cannot be regenerated, the reaction stalls. In aerobic conditions, NADH is oxidized back to NAD+ through the electron transport chain. In anaerobic conditions, fermentation pathways regenerate NAD+ to allow glycolysis to continue.

Substrate-level phosphorylation occurs when high-energy phosphate groups are directly transferred from substrate molecules to ADP, forming ATP. In the pay-off phase, phosphoglycerate kinase transfers a phosphate from 1,3-bisphosphoglycerate to ADP, and pyruvate kinase transfers a phosphate from phosphoenolpyruvate to ADP, each producing one ATP molecule.

The pay-off phase produces four ATP, two NADH, and two pyruvate molecules from two G3P molecules. After accounting for the two ATP consumed in the preparatory phase, glycolysis yields a net of two ATP, two NADH, and two pyruvate. Pyruvate can then proceed to the citric acid cycle or be converted through alternative metabolic pathways.

Phosphoglycerate mutase is an isomerase that moves the phosphate group from the third carbon to the second carbon of the sugar molecule, converting 3-phosphoglycerate into 2-phosphoglycerate. This structural rearrangement is necessary to position the phosphate group for the subsequent dehydration reaction catalyzed by enolase.