Continuous Sirtuin/HDAC (histone deacetylase) activity assay using thioamides as PET (Photoinduced Electron...

Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the ε-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted "super-substrates" with catalytic constants of up to 15,000,000 M^-1s^-1 for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD⁺ has IC₅₀ values below 200 pM.