In glycolysis, two molecules of ATP are consumed during two phosphorylation reactions to form fructose 1,6− biphosphate. In return four molecules of ATP are produced by substrate level phosphorylation (conversion of 1,3 -biphosphoglycerate to 3 -phosphoglycerate and phosphoenol pyruvate to pyruvate). Two molecules of NADH2 are formed at the time of oxidation of glyceraldehyde 3 -phosphate to 1,3 -biphosphoglycerate. The net reaction of glycolysis is as follows: Glucose+2NAD+2ADP+2H3PO4→2Pyruvate+2NADH+2H++2ATP
Each NADH is equivalent to 3ATP , so the net gain in glycolysis is 8ATP .