Role of NAD+ in the Deacetylase Activity of the SIR2-like Proteins

In this report we describe the role of NAD+ in the deacetylation reaction catalyzed by the SIR2 family of enzymes. We first show that the products of the reaction detected by HPLC analysis are ADP-ribose, nicotinamide, and a deacetylated peptide substrate. These products are in a 1:1:1 molar ratio, indicating that deacetylation involves the hydrolysis of one NAD+ to ADP-ribose and nicotinamide for each acetyl group removed. Three results suggest that deacetylation requires an enzyme–ADP-ribose intermediate. First, the enzyme can promote an NAD+ ⇔ nicotinamide exchange reaction that depends on an acetylated substrate. Second, a non-hydrolyzable NAD+ analog is a competitive inhibitor of the enzyme, and, third, nicotinamide shows product inhibition of deacetylase activity.