See, e.g., Example 2 for a non-limiting example of those methods. In some embodiments, the isolation and/or quantitation of 2′/3′-O-acetyl-ADP-ribose can be advantageously simplified by using radiolabeled NAD + such that the 2′/3′-O-acetyl-ADP-ribose is radiolabeled after the peptide is deacetylated. Preferably, the 2′/3′-O-acetyl-ADP-ribose is purified chromatographically, for example by HPLC. The 2′/3′-O-acetyl-ADP-ribose can be identified and purified from the reaction mixture. Preferably, the 2′/3′-O-acetyl-ADP-ribose is prepared using a Sir2 enzyme, by combining the Sir2 enzyme with NAD + and an acetylated peptide substrate of the Sir2 in a reaction mixture under conditions and for a time sufficient to deacetylate the peptide. The 2′/3′-O-acetyl-ADP-ribose product can be prepared by any of a number of chemical or enzymatic methods in the art. These uses for 2′/3′-AADPR are further described below. The 2′/3′-AADPR product is also useful for, inter alia, testing for inhibitors of Sir2 enzymes, for determining and quantifying Sir2 activity in a composition, and for inhibiting Sir2 enzymes. Because Sir2 uses metabolically valuable NAD + to produce 2′/3′-AADPR, and because the product 2′/3′-AADPR is metabolically unstable, the skilled artisan would understand that the product is likely useful as initiators of signaling pathways. The 2′/3′-AADPR has not been previously described. This is contrary to the previously held belief that the product of the reaction is &bgr -1′-AADPR 15,16. Thus, the Sir2 reaction ultimately causes the production of 2′/3′-O-acetyl-ADP-ribose (“2′/3′-AADPR”- FIG. The 2′ acetyl group can transfer to the 3′ hydroxyl until an equilibrium is formed between the 2′ and 3′ forms. The first discovery is the determination that a product of the reaction of a Sir2 with an acetylated peptide and NAD + is 2′-O-acetyl-ADP-ribose. The present invention is based on two discoveries. A cleft is proposed to bind the acetyl-lysine side chain of substrate proteins in proximity to the C40 of the bound NAD +, providing substrate organization for acetyl group transfer between the peptide side chain and NAD +18. The structure was interpreted in the context of a catalytic mechanism that produces &bgr -1′-AADPR 18.
A crystal structure of a Sir2p homolog from Archaeoglobus fulgidus called Sir2-Af1 was recently determined with NAD + bound at the active site.
This unusual requirement for NAD + is stoichiometric 14 and generates a novel product originally proposed to be &bgr -1′-AADPR 15,16 or possibly 2′-AADPR 16,17. Sir2 enzymes are homologs of the bacterial enzyme cobB, a phosphoribosyltransferase 13, which led to the finding that Sir2p employs NAD + as a co-substrate in deacetylation reactions 10,14,12. In yeast, these proteins form complexes with other proteins to silence chromatin 3-6 by accessing histones 7,8 and deacetylating them 9-12. The Sir2p-like enzymes are broadly conserved from bacteria to humans 1,2.
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