Sir2 protein deacetylases catalyze nicotinamide adenine dinucleotide (NAD+)-dependent deacetylation of lysine residues with the stoichiometric production of nicotinamide and O-acetyl-ADP-ribose (Figure 1). Sir2 proteins (or sirtuins) have been implicated in a variety of biological processes such as lifespan extension, neurodegeneration, fatty acid metabolism, apoptosis, and gene transcription (Blander and Guarente, 2004). Because of their requirement for NAD+, a key metabolic intermediate, sirtuins provide a unique link between cellular metabolic pathways and biological processes controlled through reversible protein acetylation. Pharmaceuticals that modulate the activity of Sir2 deacetylases may be effective in the treatment of cancer, diabetes, ageing, and neurodegeneration. Accordingly, understanding the details of substrate binding and catalysis are critical steps toward realizing these goals.