Sir2 deacetylases exhibit nucleophilic participation of acetyl-lysine in NAD+ cleavage. J Am Chem Soc 2007 May 09;129(18):5802-3
Date
04/19/2007Pubmed ID
17439123Pubmed Central ID
PMC2568996DOI
10.1021/ja070162wScopus ID
2-s2.0-34248595983 (requires institutional sign-in at Scopus site) 66 CitationsAbstract
Sir2 deacetylases (sirtuins) couple the deacetylation of lysine residues with conversion of NAD+ to O-acetyl-ADP-ribose (OAADPr) and nicotinamide. Sirtuins are potential targets for the treatment of diabetes, ageing, cancer, and neurodegenerative diseases. The most debated portion of the chemical mechanism is the initial catalytic step that forms nicotinamide and the O-alkylamidate intermediate. Here, utilizing a series of acetyl-lysine analogs that differ greatly in the electron-withdrawing nature of the substituents, we present evidence that the nucleophilicity of the acetyl-oxygen is directly tied to nicotinamide-ribosyl bond cleavage, consistent with an SN2-like mechanism for the initial step. The log of the rate of nicotinamide formation, which varied over 5-orders of magnitude, was plotted versus the inductive Taft constant, σ*, revealing a linear-free energy relationship with a steep negative slope (ρ* = -1.9). In addition, most acetyl-lysine analogs exhibited Kd values that were as low or lower than that of acetyl-lysine, indicating the diminished reactivity was due to chemistry and not substrate binding. Facile nicotinamide exchange was observed with the acetyl substrate (Vmax = 2.9 ± 0.2 s-1; Km = 406 ± 70 μM) but >400-fold slower exchange rates were observed with the fluorinated analogs. All analogs were converted to their corresponding O-acetyl-ADP-ribose analogs at a steady-state turnover rate similar to the rate of nicotinamide formation. An SN2-like mechanism in Sir2 deacetylases is unusual, as other examples of enzymatic nicotinamide-ribosyl bond cleavage proceed through an oxocarbenium intermediate in an SN1-like mechanism. These results have important implications on the selective inhibition of Sir2 over other NAD+-metabolizing enzymes.
Author List
Smith BC, Denu JMAuthor
Brian C. Smith PhD Associate Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
HydrolysisKinetics
Lysine
NAD
Sirtuins