Abstract
The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD(+)-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD(+) 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' --> 3'). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD(+) 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.
Publication types
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Adenosine Diphosphate Ribose / analogs & derivatives*
-
Adenosine Diphosphate Ribose / chemical synthesis*
-
Adenosine Diphosphate Ribose / chemistry
-
Adenosine Diphosphate Ribose / metabolism
-
Amino Acid Sequence
-
Arabinose / chemistry
-
Archaeal Proteins / chemistry
-
Archaeal Proteins / genetics
-
Bacterial Proteins / chemistry
-
Bacterial Proteins / genetics
-
Deuterium Oxide / metabolism
-
Enzyme Inhibitors / chemistry
-
Fungal Proteins / chemistry
-
Fungal Proteins / genetics
-
Gene Silencing*
-
Histone Deacetylase Inhibitors
-
Histone Deacetylases / chemistry*
-
Histone Deacetylases / genetics
-
Histone Deacetylases / metabolism
-
Humans
-
Isomerism
-
Kinetics
-
Molecular Sequence Data
-
NAD / chemistry*
-
NAD / metabolism
-
Nuclear Magnetic Resonance, Biomolecular
-
O-Acetyl-ADP-Ribose
-
Oxygen Isotopes / metabolism
-
Silent Information Regulator Proteins, Saccharomyces cerevisiae*
-
Sirtuin 1
-
Sirtuin 2
-
Sirtuins
-
Substrate Specificity
-
Trans-Activators / antagonists & inhibitors
-
Trans-Activators / chemistry*
-
Trans-Activators / genetics
-
Trans-Activators / metabolism
Substances
-
Archaeal Proteins
-
Bacterial Proteins
-
Enzyme Inhibitors
-
Fungal Proteins
-
Histone Deacetylase Inhibitors
-
O-Acetyl-ADP-Ribose
-
Oxygen Isotopes
-
Silent Information Regulator Proteins, Saccharomyces cerevisiae
-
Trans-Activators
-
NAD
-
Adenosine Diphosphate Ribose
-
Arabinose
-
SIR2 protein, S cerevisiae
-
SIRT1 protein, human
-
Sirtuin 1
-
Sirtuin 2
-
Sirtuins
-
Histone Deacetylases
-
Deuterium Oxide