1243706-42-4Relevant articles and documents
Transition state of ADP-ribosylation of acetyllysine catalyzed by archaeoglobus fulgidus Sir2 determined by kinetic isotope effects and computational approaches
Cen, Yana,Sauve, Anthony A.
, p. 12286 - 12298 (2010)
Sirtuins are protein-modifying enzymes distributed throughout all forms of life. These enzymes bind NAD+, a universal metabolite, and react it with acetyllysine residues to effect deacetylation of protein side chains. This NAD+-dependent deacetylation reaction has been observed for sirtuin enzymes derived from archaeal, eubacterial, yeast, metazoan, and mammalian species, suggesting conserved chemical mechanisms for these enzymes. The first chemical step of deacetylation is the reaction of NAD+ with an acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide. In this manuscript, the transition state for the ADP-ribosylation of acetyllysine is solved for an Archaeoglobus fulgidus sirtuin (Af2Sir2). Kinetic isotope effects (KIEs) were obtained by the competitive substrate method and were [1N-15N] = 1.024(2), [1′N-14C] = 1.014(4), [1′N- 3H] = 1.300(3), [2′N-3H] = 1.099(5), [4′N-3H] = 0.997(2), [5′N- 3H] = 1.020(5), [4′N-18O] = 0.984(5). KIEs were calculated for candidate transition state structures using computational methods (Gaussian 03 and ISOEFF 98) in order to match computed and experimentally determined KIEs to solve the transition state. The results indicate that the enzyme stabilizes a highly dissociated oxocarbenium ionlike transition state with very low bond orders to the leaving group nicotinamide and the nucleophile acetyllysine. A concerted yet highly asynchronous substitution mechanism forms the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction.
