| Sir2 enzymes, or sirtuins, are an ancient family of NAD+ dependent protein deacetylases and ADP ribosyltransferases. Sir2 enzymes regulate many biological processes including aging, transcriptional silencing, DNA repair, apoptosis, neurodegeneration, and DNA recombination. Sir2 enzymes consume one molecule of NAD+ to deacetylate an acetyl lysine on a protein substrate and yield lysine, 0-acetyl-ADP ribose, and nicotinamide. Structural studies have detailed how Sir2 enzymes bind substrates to promote the deacetylation reaction, but prior to my work, no structure existed of a Sir2 enzyme bound to a reaction intermediate. Structural and biochemical data presented in this thesis describe a Sir2 enzyme bound to an acetylated peptide and a dissociated NAD+ analogue and Sir2 bound to an S-alkylamidate intermediate, which is analogous to the naturally occurring O-alkylamidate intermediate. Sir2 mediated ADP ribosylation is a known activity for many Sir2 enzymes. However, the mechanism of Sir2 ADP ribosylation is not well defined. Structural, biochemical, and biophysical data presented here describe a mechanism for acetyl lysine dependent Sir2 mediated ribosylation. |
