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Acetyl-coenzyme A (Acetyl-CoA) is a membrane-impermeant molecule constituted by an acetyl moiety (CH3CO) linked to coenzyme A (CoA), a derivative of vitamin B5 and cysteine, through a thioester bond. As thioester bonds are energy rich, the chemical structure of acetyl-CoA facilitates the transfer of the acetyl moiety to a variety of acceptor molecules, including amino groups on proteins.
In most mammalian cells, Acetyl-coenzyme A (Acetyl-CoA) is predominantly generated in the mitochondrial matrix by various metabolic circuitries, namely glycolysis, β-oxidation, and the catabolism of branched amino acids. Cytosolic Acetyl-coenzyme A is the precursor of multiple anabolic reactions that underlie the synthesis of fatty acids and steroids, as well as specific amino acids including glutamate, proline, and arginine.

Mice deprived of food (but with access to water ad libitum) for 24 hr exhibit a significant reduction in total Acetyl-coenzyme A (Acetyl-CoA) levels in several organs, including the heart and muscles, corresponding to a decrease in protein acetylation levels. However, the same experimental conditions have no major effects on Acetyl-coenzyme A concentrations in the brain and actually increase hepatic Acetyl-coenzyme A and protein acetylation levels. Ethanol intake augments Acetyl-coenzyme A levels in hepatic mitochondria.