title: A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.
abstract: Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1-6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M(-1)s(-1). These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM).
authors.affiliation: Department of Enzymology, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 0610 Halle (Saale), Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Schuster
authors.affiliation: Department of Enzymology, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 0610 Halle (Saale), Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Roessler
authors.affiliation: Department of Enzymology, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 0610 Halle (Saale), Germany.
authors.forename: Marat
authors.initials: M
authors.lastname: Meleshin
authors.affiliation: Department of Enzymology, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 0610 Halle (Saale), Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Zimmermann
authors.affiliation: Department of Enzymology, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 0610 Halle (Saale), Germany.
authors.forename: Zeljko
authors.initials: Z
authors.lastname: Simic
authors.affiliation: Department of Biochemistry, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Kambach
authors.affiliation: Department of Enzymology, joint research project gFP5, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 0610 Halle (Saale), Germany.
authors.forename: Cordelia
authors.initials: C
authors.lastname: Schiene-Fischer
authors.affiliation: Department of Biochemistry, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany.
authors.forename: Clemens
authors.initials: C
authors.lastname: Steegborn
authors.affiliation: IVBMB, University of Zurich-Irchel, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Michael O
authors.initials: MO
authors.lastname: Hottiger
authors.affiliation: Department of Enzymology, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 0610 Halle (Saale), Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Schutkowski
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D006801:Humans; D037761:Sirtuins / Q000032:analysis* / Q000037:antagonists & inhibitors*
chemical_list: D004791:Enzyme Inhibitors; D037761:Sirtuins