Wolfson Noah A, Pitcairn Carol Ann, Sullivan Eric D, Joseph Caleb G, Fierke Carol A
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Interdepartmental Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109, USA.
Anal Biochem. 2014 Jul 1;456:61-9. doi: 10.1016/j.ab.2014.03.012. Epub 2014 Mar 25.
Histone deacetylases catalyze the hydrolysis of an acetyl group from post-translationally modified acetyl-lysine residues in a wide variety of essential cellular proteins, including histones. Because these lysine modifications can alter the activity and properties of affected proteins, aberrant acetylation/deacetylation may contribute to disease states. Many fundamental questions regarding the substrate specificity and regulation of these enzymes have yet to be answered. Here, we optimize an enzyme-coupled assay to measure low micromolar concentrations of acetate, coupling acetate production to the formation of NADH (nicotinamide adenine dinucleotide, reduced form) that is measured by changes in either absorbance or fluorescence. Using this assay, we measured the steady-state kinetics of peptides representing the H4 histone tail and demonstrate that a C-terminally conjugated methylcoumarin enhances the catalytic efficiency of deacetylation catalyzed by cobalt(II)-bound histone deacetylase 8 [Co(II)-HDAC8] compared with peptide substrates containing a C-terminal carboxylate, amide, and tryptophan by 50-, 2.8-, and 2.3-fold, respectively. This assay can be adapted for a high-throughput screening format to identify HDAC substrates and inhibitors.
组蛋白脱乙酰酶催化从多种重要细胞蛋白（包括组蛋白）中经翻译后修饰的乙酰赖氨酸残基上水解乙酰基。由于这些赖氨酸修饰可改变受影响蛋白的活性和特性，异常的乙酰化/去乙酰化可能导致疾病状态。关于这些酶的底物特异性和调控的许多基本问题尚未得到解答。在此，我们优化了一种酶联测定法，以测量低微摩尔浓度的乙酸盐，将乙酸盐的产生与NADH（烟酰胺腺嘌呤二核苷酸，还原形式）的形成相偶联，通过吸光度或荧光的变化来测量NADH。使用该测定法，我们测量了代表H4组蛋白尾巴的肽段的稳态动力学，并证明与含有C末端羧酸盐、酰胺和色氨酸的肽底物相比，C末端共轭甲基香豆素可使钴（II）结合的组蛋白脱乙酰酶8 [Co（II）-HDAC8]催化的去乙酰化催化效率分别提高50倍、2.8倍和2.3倍。该测定法可适用于高通量筛选形式，以鉴定HDAC底物和抑制剂。
