ATP is generated by substrate-level phosphorylation by high-energy compounds such as 13-bisphosphoglycerate and phosphoenolpyruvateGlycolysis is used by all cells in the body for energy generation. The ATP made during glycolysis is generated by substrate-level phosphorylation. Atp Production Of One Glucose In glycolysis 4 ATP are generated through substrate-level phosphorylation. . The energy payoff phase of glycolysis consists of five additional steps and results in the formation of four ATP two NADH H and two pyruvate molecules. Substrate-level phosphorylation is a type of metabolic reaction that results in the formation of Adenosine Triphosphate ATP or Guanosine Triphosphate GTP by the direct transfer and donation of a phosphoiy1 PO_3 group to Adenosine Diphosphate ADP or Guanosine Diphosphate GDP from a phosphorylated reactive intermediate. Biology textbooks often state that 38 ATP molecules can be made per oxidized glucose molecule du