title: An enzyme-coupled assay measuring acetate production for profiling histone deacetylase specificity.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Noah A
authors.initials: NA
authors.lastname: Wolfson
authors.affiliation: Interdepartmental Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Carol Ann
authors.initials: CA
authors.lastname: Pitcairn
authors.affiliation: Interdepartmental Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Sullivan
authors.affiliation: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Caleb G
authors.initials: CG
authors.lastname: Joseph
authors.affiliation: 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; Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA; Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address: fierke@umich.edu.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D000085:Acetates / Q000378:metabolism*; D000595:Amino Acid Sequence; D055162:Biocatalysis; D057075:Enzyme Assays / Q000379:methods*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000085:Acetates; D056572:Histone Deacetylase Inhibitors; D009842:Oligopeptides; D009243:NAD; D006655:Histone Deacetylases
keywords: Acetate detection assay; Catalysis; Fluorescence; Histone deacetylase 8 (HDAC8)