| Protein post-translational modifications provide a wealth of complexity and diversity that result in profound influences on the structure and function of proteins in cells. The power of these modifications lies in their ability to be dynamically regulated by a set of highly conserved enzymes, creating an ever changing environment within the context of the cell. In the nucleus, chromatin is a complex macromolecule that provides a scaffold for the packaging of DNA. The modifications present on the contained histone tails dictate the transcriptional state of the underlying DNA by their ability to recruit proteins and protein complexes. Lysine specific demethylase 1 (LSD1) is a member of the amine oxidase superfamily and utilizes a non-covalently bound FAD cofactor in the oxidative removal of methyl groups, specifically from Lys-4 of histone H3, and is a component of various transcriptional repressor complexes.;Herein, we report the mechanism-based inactivation of LSD1 by the propargylamine, cyclopropylamine, 3-chloroallylamine, and hydrazino functionalities. Kinetic and mechanistic analysis utilizing in vitro enzyme assays, MALDI-TOF mass spectrometry, UV-Vis spectroscopy, NMR spectroscopy, and X-ray crystallography have allowed us to confidently assign the mechanism of inactivation as the formation of inactivator-FAD adducts. We further demonstrate the utility of these peptide-based inactivators for the discovery of novel flavin-dependent demethylases by successfully "fishing" for endogenous LSD1 from HeLa cell nuclear extracts. |
