title: A Novel Continuous Assay for the Deacylase Sirtuin 5 and Other Deacetylases.
abstract: Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes like control of metabolic pathways, DNA repair, and stress response. Modulators of sirtuin activity are needed as tools for uncovering the biological function of these enzymes and as potential therapeutics. Systematic discovery of such modulators is hampered by the lack of efficient and simple continuous activity assays running at low sirtuin concentrations in microtiter plates. Here we describe an improved continuous sirtuin 5 assay based on the coupling of the sirtuin reaction to a proteolytic cleavage using internally fluorescence-quenched substrates. Systematic optimization of a carbamoyl phosphate synthetase 1 derived, glutarylated peptide yielded a Sirt5 substrate with k(cat)/K(M) value of 337,000 M(-1) s(-1), which represents the best sirtuin substrate described so far. These extraordinary substrate properties allowed reliable determination of Ki values for different inhibitors in the presence of only 10 nM sirtuin in microtiter plate format. Assay conditions could be transferred effectively to other lysine deacetylases, like sirtuin 2 and sirtuin 3, which now enables more efficient development of sirtuin targeting drugs.
authors: Department of Enzymology, Martin-Luther-University Halle-Wittenberg , Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
authors: Claudia
authors: C
authors: Roessler
authors: Department of Enzymology, Martin-Luther-University Halle-Wittenberg , Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
authors: Christian
authors: C
authors: Tüting
authors: Department of Enzymology, Martin-Luther-University Halle-Wittenberg , Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
authors: Marat
authors: M
authors: Meleshin
authors: Department of Biochemistry, University of Bayreuth , Universitaetsstrasse 30, 95447 Bayreuth, Germany.
authors: Clemens
authors: C
authors: Steegborn
authors: Department of Enzymology, Martin-Luther-University Halle-Wittenberg , Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany.
authors: Mike
authors: M
authors: Schutkowski
mesh_terms: D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry*; D057075:Enzyme Assays / Q000379:methods; D056572:Histone Deacetylase Inhibitors / Q000737:chemistry; D006655:Histone Deacetylases / Q000737:chemistry*; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry*; D059748:Proteolysis; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D056572:Histone Deacetylase Inhibitors; D009842:Oligopeptides; D037761:Sirtuins; D006655:Histone Deacetylases; D002222:Carbamoyl-Phosphate Synthase (Ammonia)