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, aging, 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 analogues 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 S(N)2-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, sigma*, revealing a linear free energy relationship with a steep negative slope (rho* = -1.9). In addition, most acetyl-lysine analogues exhibited K-d 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 (V-max = 2.9 +/- 0.2 s(-1); K-m = 406 +/- 70 mu M), but > 400-fold slower exchange rates were observed with the fluorinated analogues. All analogues were converted to their corresponding O-acetyl-ADP-ribose analogues at a steady-state turnover rate similar to the rate of nicotinamide formation. An S(N)2-like mechanism in Sir2 deacetylases is unusual, as other examples of enzymatic nicotinamide-ribosyl bond cleavage proceed through an oxocarbenium intermediate in an S(N)1-like mechanism. These results have important implications on the selective inhibition of Sir2 over other NAD(+)-metabolizing enzymes.