sirt5 deacylation activities show differential sensitivities to nicotinamide inhibitionsirt5显示敏感性差烟酰胺抑制脱酰作用的活动.pdfVIP

Sirt5 Deacylation Activities Show Differential Sensitivities to Nicotinamide Inhibition 1. 1. 1 1 Frank Fischer , Melanie Gertz , Benjamin Suenkel , Mahadevan Lakshminarasimhan , 2 1 Mike Schutkowski , Clemens Steegborn * 1 Department of Biochemistry and Research Center for Bio-Macromolecules, University of Bayreuth, Bayreuth, Germany, 2 Department of Enzymology, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle, Germany Abstract Sirtuins are protein deacylases regulating metabolism and aging processes, and the seven human isoforms are considered attractive therapeutic targets. Sirtuins transfer acyl groups from lysine sidechains to ADP-ribose, formed from the cosubstrate NAD+ by release of nicotinamide, which in turn is assumed to be a general Sirtuin inhibitor. Studies on Sirtuin regulation have been hampered, however, by shortcomings of available assays. Here, we describe a mass spectrometry– based, quantitative deacylation assay not requiring any substrate labeling. Using this assay, we show that the deacetylation activity of human Sirt5 features an unusual insensitivity to nicotinamide inhibition. In contrast, we find similar values for Sirt5 and Sirt3 for the intrinsic NAD+ affinity as well as the apparent NAD+ affinity in presence of peptide. Structure comparison and mutagenesis identify an Arg neighboring to the Sirt5 nicotinamide binding pocket as a mediator of nicotinamide resistance, and statistical sequence analyses along with testing further Sirtuins reveal a network of coevolved residues likely defining a nicotinamide-insensitiv