We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetylCoA synthetase activity of AceCS2. This report identifies the first acetylated substrate protein of SIRT3. Our findings show that a mammalian sirtuin directly controls the activity of a metabolic enzyme by means of reversible lysine acetylation. Because the activity of a bacterial ortholog of AceCS2, called ACS, is controlled via deacetylation by a bacterial sirtuin protein, our observation highlights the conservation of a metabolic regulatory pathway from bacteria to humans.eversible lysine acetylation is a highly regulated posttranslational protein modification, which is controlled by protein deacetylases and acetyltransferases (1, 2). Although the importance of reversible lysine acetylation of nuclear nonhistone and histone proteins is well established, the role of protein modification by reversible lysine acetylation in mitochondria is unknown.The nicotinamide (NAM) adenine dinucleotide (NAD ϩ )-dependent deacetylase silent information regulator 2 (sir2) is an important mediator of longevity in response to caloric restriction signals in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster (3-9). Seven mammalian Sir2 homologs (SIRT1-7) are known (10-13). The recent discovery that SIRT3, SIRT4, and SIRT5 are found in mitochondria (14-17) suggests the existence of mitochondrial sirtuin substrate proteins. Results and DiscussionAs a strategy to identify lysine-acetylated mitochondrial proteins that could be targeted by SIRT3, -4, or -5, we searched for human proteins with sequence similarity to the region surrounding the acetylated lysine residue found in acetyl-CoA synthetase (ACS) from Salmonella enterica. We chose the acetyl-lysine-containing region of S. enterica ACS, because it is a known substrate for the sirtuin CobB (18,19). In a second step, human proteins showing high similarity were analyzed for their mitochondrial localization probability by using MITOPROT II and PREDOTAR Ver.