Investigating the ADP-ribosyltransferase activity of sirtuins with NAD analogues and 32P-NAD.
Protein ADP-ribosyltransferases catalyze the transfer of adenosine diphosphate ribose (ADP-ribose) 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 32P-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 kcat/Km values of the deacetylation activity of sirtuins, the observed rate constant of sirtuin-dependent ADP-ribosyltion is 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.
Du
Jintang
J
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Jiang
Hong
H
Lin
Hening
H
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000107:Acetylation / Q000187:drug effects; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry / Q000378:metabolism; D000480:Alkynes / Q000378:metabolism; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D004167:Diphtheria Toxin / Q000494:pharmacology; D006367:HeLa Cells; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D009243:NAD / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D037761:Sirtuins / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects
D000480:Alkynes; D004167:Diphtheria Toxin; D006657:Histones; D020652:Peptide Elongation Factor 2; D010761:Phosphorus Radioisotopes; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; D037761:Sirtuins; D006655:Histone Deacetylases