High-throughput screening of histone deacetylases and determination of kinetic parameters using fluorogenic assays

Histone deacetylases (HDACs) are ubiquitous enzymes that cleave post -transla-tional e-N-acyllysine modifications. The continued identification of diverse acyl modifications at lysine residues in proteins has resulted in discovery of new insight into the biological roles of these enzymes. Here, we describe a fluoro-genic high-throughput screening protocol to identify deacylase activities. We describe the careful optimization of continuous, coupled enzyme assays, which provide efficient determination of kinetic parameters. These techniques can facilitate inhibitor assay design and provide fundamental understanding of HDAC biochemistry. For complete details on the use and execution of this protocol, please refer to Moreno-Yruela et al. (2018).

Automated summary

Histone deacetylases (HDACs) are enzymes that cleave post-translational e-N-acyllysine modifications. A high-throughput screening protocol is described to identify deacylase activities, with careful optimization of continuous enzyme assays to determine kinetic parameters efficiently. These techniques can aid in inhibitor assay design and provide fundamental understanding of HDAC biochemistry.