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The generation of ATP directly from an intermediate substrate during cellular respiration occurs through substrate-level phosphorylation. This process involves the transfer of a phosphate group from a phosphorylated intermediate directly to ADP, forming ATP. This mechanism is distinct from oxidative phosphorylation, where ATP is produced indirectly through a series of reactions in the electron transport chain and chemiosmosis.

Substrate-level phosphorylation primarily takes place during glycolysis and the Krebs cycle, where specific enzymes facilitate the transfer of phosphates to ADP from high-energy substrate molecules. For example, in glycolysis, one of the steps involves the enzyme phosphoglycerate kinase transferring a phosphate from 1,3-bisphosphoglycerate to ADP, thereby forming ATP.

This direct synthesis of ATP during metabolic pathways highlights the importance of intermediary substrates in energy production. Understanding substrate-level phosphorylation is crucial when studying pathways of cellular respiration and the overall energy balance in cells.