Center for Biopharmaceuticals and Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.
STAR Protoc. 2021 Feb 3;2(1):100313. doi: 10.1016/j.xpro.2021.100313. eCollection 2021 Mar 19.
Histone deacetylases (HDACs) are ubiquitous enzymes that cleave post-translational ε--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 fluorogenic 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).
组蛋白去乙酰化酶（HDACs）是一种普遍存在的酶，能够切割翻译后 ε--酰化赖氨酸修饰。在蛋白质赖氨酸残基上不断发现各种酰基修饰，这使得人们对这些酶的生物学作用有了新的认识。在这里，我们描述了一种荧光高通量筛选方案，以鉴定去乙酰化酶的活性。我们描述了连续偶联酶测定的仔细优化，这为动力学参数的有效测定提供了条件。这些技术可以促进抑制剂测定的设计，并提供对 HDAC 生物化学的基本理解。有关该方案使用和执行的完整详细信息，请参阅 Moreno-Yruela 等人。（2018 年）。
