Sirtuins are implicated in the regulation of diverse cellular processes, such as stress resistance, metabolic control, apoptotic signaling and chromatin regulation. Sirtuins regulate these processes by utilizing NAD+ to accomplish deacetylation and ADP-ribosylation reactions on a variety of cellular proteins. The catalytic mechanisms for sirtuin catalyzed reactions are under investigation in our laboratory. The deacetylation reaction is proposed to be initiated by ADP-ribosylation of acetyllysine to form a peptidylimidate intermediate with alpha-stereochemistry (Sauve et al Biochemistry 2001, 40(51):15456-63). Several chemistries have been attributed to the peptidylimidate intermediate, including nicotinamide base exchange and deacetylation reactions. Although the peptidyl intermediate and its chemistries have considerable experimental support, chemical characterization of this intermediate (as well as other intermediates of sirtuin reaction pathways) has proven challenging. ADP-ribosylation reactions on protein substrates independent of deacetylation have also been reported. The mechanism and reaction products for this latter reaction have not been elucidated. Several sirtuins studied within our lab have provided distinct mechanistic examples that provide insights into the NAD+ deacetylation reaction mechanism and on reports of ADP-ribosylation observed for sirtuin enzymes.