Substrate-level phosphorylation

[2] The high energy bonds between the phosphate groups can be broken to power a variety of reactions used in all aspects of cell function.

[3] Substrate-level phosphorylation occurs in the cytoplasm of cells during glycolysis and in mitochondria either during the Krebs cycle or by MTHFD1L (EC 6.3.4.3), an enzyme interconverting ADP + phosphate + 10-formyltetrahydrofolate to ATP + formate + tetrahydrofolate (reversibly), under both aerobic and anaerobic conditions.

ATP can be generated by substrate-level phosphorylation in mitochondria in a pathway that is independent from the proton motive force.

In the matrix there are three reactions capable of substrate-level phosphorylation, utilizing either phosphoenolpyruvate carboxykinase or succinate-CoA ligase, or monofunctional C1-tetrahydrofolate synthase.

Mitochondrial phosphoenolpyruvate carboxykinase is thought to participate in the transfer of the phosphorylation potential from the matrix to the cytosol and vice versa.

The potential energy stored as an electrochemical gradient of protons (H+) across the inner mitochondrial membrane is required to generate ATP from ADP and Pi (inorganic phosphate molecule), a key difference from substrate-level phosphorylation.