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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 phosphoryl (PO3) group to adenosine diphosphate (ADP) or guanosine diphosphate (GDP) from a phosphorylated reactive intermediate. Note that the phosphate group does not have to come directly from the substrate. By convention, the phosphoryl group that is transferred is referred to as a phosphate group.
An alternative way to create ATP is through oxidative phosphorylation, which takes place during the process of cellular respiration, in addition to the substrate-level phosphorylation that occurs during glycolysis and the Krebs cycle. During oxidative phosphorylation, NADH is oxidized to NAD+, yielding 2.5 ATPs, and FADH2 yields 1.5 ATPs when it is oxidized. Oxidative phosphorylation uses an electrochemical or chemiosmotic gradient of protons (H+) across the inner mitochondrial membrane to generate ATP from ADP, which is a key difference from substrate-level phosphorylation.
Unlike oxidative phosphorylation, oxidation and phosphorylation are not coupled in the process of substrate-level phosphorylation, although both types of phosphorylation result in the formation of ATP, and reactive intermediates are most often gained in course of oxidation processes in catabolism. However, usually most of the ATP is generated by oxidative phosphorylation in aerobic or anaerobic respiration. Substrate-level phosphorylation serves as fast source of ATP independent of external electron acceptors and respiration. This is the case for example in human erythrocytes, which have no mitochondria, and in the muscle during oxygen depression.
The main part of substrate-level phosphorylation occurs in the cytoplasm of cells as part of glycolysis and in mitochondria as part of the Krebs Cycle under both aerobic and anaerobic conditions. In the pay-off phase of glycolysis, two ATP are produced by substrate-level phosphorylation: two and only two 1,3-bisphosphoglycerate are converted to 3-phosphoglycerate by transferring a phosphate group to ADP by a kinase; two phosphoenolpyruvate are converted to pyruvate by the transfer of their phosphate groups to ADP by another kinase. The first reaction occurs after the generation of 1,3-bisphosphoglycerate from 3-phosphoglyceraldehyde and an organic phosphate via a dehydrogenase. ATP is generated in a following separate step (key difference from oxidative phosphorylation) by transfer of the high-energy phosphate on 1,3-bisphosphoglycerate to ADP via the enzyme phosphoglycerate kinase, generating 3-phosphoglycerate. As ATP is formed of a former inorganic phosphate group, this step leads to the energy yield of glycolysis. The second substrate-level phosphorylation occurs later by means of the reaction of phosphenolpyruvate (PEP) to pyruvate via the pyruvate kinase. This reaction regenerates the ATP that has been used in the preparatory phase of glycolysis to activate glucose to glucose-6-phosphate and fructose-6-phosphate to fructose-1,6-bisphosphate, respectively.
ATP can be generated by substrate-level phosphorylation in the mitochondrial matrix, a pathway that is independent from the protonmotive force, pmf. In the mitochondrial matrix there are two reactions capable of substrate-level phosphorylation: the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK), and the succinate-CoA ligase (SUCL or succinate thiokinase or succinyl-CoA synthetase). Mitochondrial PEPCK is thought to participate in the transfer of the phosphorylation potential from the matrix to cytosol and vice versa. Succinate-CoA ligase is a heterodimer, being composed of an invariant alpha subunit, and a substrate-specific beta subunit, encoded by either SUCLA2 or SUCLG2. This dimer combination results in either an ADP-forming succinate-CoA ligase (A-SUCL, EC 184.108.40.206) or a GDP-forming succinate-CoA ligase (G-SUCL, EC 220.127.116.11). The ADP-forming succinate-CoA ligase is potentially the only matrix enzyme generating ATP in the absence of a pmf, capable of maintaining matrix ATP levels under energy-limited conditions, such as transient hypoxia.
Another form of substrate-level phosphorylation is also seen in working skeletal muscles and the brain. Phosphocreatine is stored as a readily available high-energy phosphate supply, and the enzyme creatine phosphokinase transfers a phosphate from phosphocreatine to ADP to produce ATP. Then the ATP releases giving chemical energy.
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