Kinetic Analysis of HDAC- and Sirtuin-Mediated Deacylation on Chemically Defined Histones and Nucleosomes.

Histone deacetylases (HDACs) and sirtuins (SIRTs) play essential roles in regulating chromatin structure and gene expression by catalyzing the removal of acyl groups from histone lysine residues. Accurate characterization of their deacetylation kinetics is critical for understanding their enzymatic mechanisms and for guiding inhibitor or activator development. Given the complexity of enzyme-nucleosome core particle (NCP) interactions, including the influence of histone composition, post-translational modifications, and DNA context, NCP-based assays provide a more physiologically relevant platform than those performed on peptide or free histone substrates. Here, we present optimized protocols for assessing HDAC and SIRT deacetylation kinetics using NCP substrates, including determination of Michaelis-Menten parameters, evaluation of inhibitor and activator potency (IC50 or EC50), and instructions for ensuring assay reproducibility. These methods enable robust comparison of small-molecule modulators under conditions that better mimic the native chromatin environment, supporting both mechanistic studies and drug discovery efforts. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Assay of HDAC complex or SIRT deacylation on nucleosome substrates Basic Protocol 2: Assay of HDAC complex or SIRT deacylation on free histone proteins Basic Protocol 3: KM measurement for deacylation assay on nucleosome or cofactor Basic Protocol 4: Assessment of deacylation inhibitor or activator effects on NCP substrates.