Substrate-level Phosphorylation

Substrate-level phosphorylation is a mechanism for producing ATP or another nucleoside triphosphate, such as GTP, by coupling its formation to an energy-releasing metabolic reaction involving a high-energy substrate. Unlike oxidative phosphorylation, it does not require an electron-transport chain or a proton gradient across a membrane .^1,2

In glycolysis, substrate-level phosphorylation occurs when enzymes transfer a phosphate group from a phosphorylated metabolic intermediate to ADP. Phosphoglycerate kinase transfers phosphate from 1,3-bisphosphoglycerate to ADP, while pyruvate kinase transfers phosphate from phosphoenolpyruvate to ADP. Both reactions produce ATP .¹

Substrate-level phosphorylation also occurs in the citric acid cycle. During conversion of succinyl-CoA to succinate, succinyl-CoA synthetase uses the favorable free-energy change of the reaction to produce GTP or ATP, depending on the enzyme isoform and tissue. This reaction proceeds through phosphorylated intermediates and does not depend directly on the mitochondrial respiratory chain .²

Substrate-level phosphorylation is therefore distinct from oxidative phosphorylation. Oxidative phosphorylation uses energy from electron transport to generate a proton gradient that drives ATP synthase. Substrate-level phosphorylation generates ATP or GTP through individual enzyme-catalyzed metabolic reactions. The mechanism itself does not require oxygen, although it can occur during aerobic metabolism .^1,2