Chemistry of Gene Silencing: The Mechanism of NAD super(+)-Dependent Deacetylation Reactions

• Published in: Biochemistry (Easton) Vol. 40; no. 51; pp. 15456 - 15463
• Main Authors: Sauve, A A, Celic, I, Avalos, J, Deng, Haiteng, Boeke, J D, Schramm, V L
• Format: Journal Article
• Language: English
• Published: 25-12-2001
• ISSN: 0006-2960
• DOI: 10.1021/bi011858j

The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD super(+)-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD super(+) are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2' and 3' regioisomers of O-acetyl ADP ribose (AADPR), formed through an alpha -1'-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2' arrow right 3'). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, super(18)O exchange, and MS methods. The mechanism of acetyl transfer to NAD super(+) includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2'-OH group on the amidate to form a 1',2'-acyloxonium species, (3) hydrolysis to 2'-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2'- and 3'-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2'- and 3'-AADPR products are candidate molecules for SIR2-initiated signaling pathways.