In this application note it is shown how the enzymatic activity of a sirtuin-type enzyme is determined using a commercial kit. Sirtuins belong to the group of histone deacetylases and deacetylate acetylated lysine residues in proteins. This activity makes these enzymes and its inhibitors interesting for the epigenetic drug discovery.
The CycLex SIRT1 assay is a peptidase coupled lysine deacetylase assay and the fluorescence signals have been measured on the CLARIOstar® microplate reader using the LVF monochromator™. Kinetic parameters Km, kcat and Vmax have been calculated for the recombinant Escherichia coli CobB enzyme; IC50 values have been determined for a known sirtuin inhibitor.