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A Novel Continuous Assay for the Deacylase Sirtuin 5 and Other Deacetylases

Title data

Roessler, Claudia ; Tüting, Christian ; Meleshin, Marat ; Steegborn, Clemens ; Schutkowski, Mike:
A Novel Continuous Assay for the Deacylase Sirtuin 5 and Other Deacetylases.
In: Journal of Medicinal Chemistry. (8 September 2015) .
ISSN 1520-4804
DOI: https://doi.org/10.1021/acs.jmedchem.5b00293

Abstract in another language

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 kcat/KM 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.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 26308971
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Clemens Steegborn
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 17 Sep 2015 06:42
Last Modified: 17 Sep 2015 06:42
URI: https://eref.uni-bayreuth.de/id/eprint/19446