The Sir2 (or sirtuin) protein family mediates lifespan, DNA repair, apoptosis, resistance to cell stress, and metabolism. However, the molecular basis for the varied phenotypes remains elusive. Most sirtuins catalyze a reaction in which the cleavage of NAD⁺ and histone/protein deacetylation are coupled to the formation of O-acetyl-ADP-ribose (OAADPr), a novel metabolite. Several reports suggest that some sirtuins are mono-ADP-ribosyltransferases, while others have suggested that these enzymes harbor both activities. Using a series of acetyl-lysine analogues within a histone H3 peptide, we have explored the catalytic pathways that lead to either protein deacetylation or protein ADP-ribosylation. The results are consistent with a deacetylation mechanism where the acetylated-lysine oxygen acts as a nucleophile, displacing nicotinamide to form an α-1'-O-alkylamidate intermediate. Next, a conserved histidine activates the 2'-hydroxyl for attack of the α-1'-O-alkylamidate. Addition of water produces the final product 2'-O-acetyl-ADP-ribose. Protein ADP-ribosylation by sirtuins appears to be a less efficient reaction that may result from nucleophiles intercepting the the α-1'-O-alkylamidate intermediate. Cellular OAADPr may function as a second messenger or a ligand/substrate for downstream protein targets. We demonstrate that the TRPM2 ion channel binds and is activated by OAADPr. We investigated whether mammalian sirtuins could directly control the activity of metabolic enzymes. We demonstrated that mammalian Acetyl-CoA Synthetases (AceCSs) are regulated by reversible acetylation and that sirtuins activate AceCSs by deacetylation. SIRT1 was the only member of seven human Sir2 homologues capable of deacetylating AceCS1 in cellular co-expression experiments. SIRT1 expression led to a pronounced increase in AceCS1-dependent fatty-acid synthesis from acetate. We also show that mitochondrial AceCS2 is regulated by acetylation, and specifically deacetylated by mitochondrial SIRT3.