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. 2007 May 9;129(18):5802-3.
doi: 10.1021/ja070162w. Epub 2007 Apr 17.

Sir2 deacetylases exhibit nucleophilic participation of acetyl-lysine in NAD+ cleavage

Brian C Smith  1 , John M Denu
Affiliations

Sir2 deacetylases exhibit nucleophilic participation of acetyl-lysine in NAD+ cleavage

Brian C Smith et al. J Am Chem Soc. .

Abstract

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.

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Figures

Figure 1
Figure 1
(Left) Single-turnover kinetics of acetyl-lysine analogs monitoring nicotinamide formation (Right) Plot of log rate of nicotinamide formation vs. the Taft σ* constant
Scheme 1
Scheme 1
Possible chemical mechanisms for initial acetyl-lysine analog attack and complete deacetylation mechanism.
See this image and copyright information in PMC

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