Investigating the ADP-ribosyltransferage Activity of Sirtuins with NAD Analogues and 32P-NAD

Protein ADP-ribosyltransferases catalyze the transfer of adenosine diphosphate ribose (ADPribose) from nicotinamide adenine dinucleotide (NAD) onto specific target proteins. Sirtuins, a class of enzymes with NAD-dependent deacetylase activity, have been reported to possess ADP-ribosyltransferase activity, too. Here we used NAD analogues and P-32-NAD to study the ADP-ribosyltransferase activity of several different sirtuins, including yeast Sir2, human SirT1, mouse SirT4, and mouse SirT6. The results showed that an alkyne-tagged NAD is the substrate for deacetylation reactions but cannot detect the ADP-ribosylation activity. Furthermore, comparing with a bacterial ADP-ribosyltransferase diphtheria toxin, the observed rate constant of sirtuin-dependent ADP-ribosylation is >5000-fold lower. Compared with the k(cat)/K-m values of the deacetylation activity of sirtuins, the observed rate constant of sirtuin-dependent ADP-ribosyltion is similar to 500 times weaker. The weak ADP-ribosylation events can be explained by both enzymatic and nonenzymatic reaction mechanisms. Combined with recent reports on several other sirtuins, we propose that the reported ADP-ribosyltransferase activity of sirtuins is likely some inefficient side reactions of the deacetylase activity and may not be physiologically relevant.