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 o f 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 AD P-ribose and nicotinamide for each acetyl group removed. Three results sugg est that deacetylation requires an enzyme-ADP-ribose intermediate. First, t he enzyme can promote an NAD(+) double left right arrow nicotinamide exchan ge reaction that depends on an acetylated substrate. Second, a non-hydrolyz able NAD(+) analog is a competitive inhibitor of the enzyme, and, third, ni cotinamide shows product inhibition of deacetylase activity. (C) 2000 Acade mic Press.