HPLC-Based Enzyme Assays for Sirtuins

Sirtuins are a class of enzymes that utilize nicotinamide adenine dinucleotide, NAD⁺, to remove various acyl groups from protein lysine residues. They have important biological functions and regulate numerous biological pathways. Small molecules that can modulate sirtuin enzymatic activities are potential therapeutic candidates to treat various human diseases. This protocol describes a high-performance liquid chromatography (HPLC)-based method to measure the enzyme kinetics for SIRT2 and SIRT6's demyristoylase activities and SIRT5's desuccinylase activity. This method uses peptide substrates that resemble physiological substrates and thus can give more reliable kinetic parameters (K _m and k _cat values) for these enzymes. The data obtained are useful for understanding the biological function of sirtuins and developing sirtuin modulators.