| Protein deacetylation and demethylation are essential processes through which cells can regulate many processes, including transcription, protein localization, and proliferation. The sirtuins are a sub-class of histone deacetylases (HDACs) that use the NAD cofactor to deacetylate acetyl-lysine residues. Here, we report the synthesis of a novel acetyl-lysine analogue, acetyl-azalysine, that incorporates a hydrazine functionality to probe protein deacetylation. We have discovered that the acetyl-azalysine modification is a good substrate for the sirtuins, but a weak substrate for HDACS, a classical HDAC. We have also indentified a novel ADP-ribose adduct, which is absolutely dependent upon the acetyl-azalysine functionality, that is a unique reaction side-product rather than the corresponding classical ADP-ribosylation modification. We explore this novel adduct using a variety of biochemical and spectroscopic methods, including the synthesis of an isotopically labeled version of acetyl-azalysine, the use of a biotinylated NAD substrate to detect the formation of the ADP-ribose adduct, mass spectrometric analysis of the product, and ultimate NMR characterization of the purified adduct. These data provide new evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed during the course of the sirtuin reactions. We were also able to synthesize the corresponding deacetylated, azalysine modification to investigate its ability to inhibit lysine-specific demethylase 1 (LSD1). We found that the azalysine modification, in the context of a peptide backbone, is a potent inhibitor of LSD1 and forms a peptide-flavin adduct that is characteristic of suicide inhibitors previously reported in the literature. |
