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Mechanistic analysis of a suicide inactivator of histone demethylase LSD1.
- Source :
-
Biochemistry [Biochemistry] 2007 Jun 12; Vol. 46 (23), pp. 6892-902. Date of Electronic Publication: 2007 May 19. - Publication Year :
- 2007
-
Abstract
- Lysine-specific demethylase 1 (LSD1) is a transcriptional repressor and a flavin-dependent amine oxidase that is responsible for the removal of methyl from lysine 4 of histone H3. In this study, we characterize the mechanism and scope of LSD1 inhibition by a propargylamine-derivatized histone H3 substrate (1). Unlike aziridinyl and cyclopropylamine-derivatized histone H3 peptide substrate analogues, compound 1 appears to covalently modify and irreversibly inactivate LSD1 with high potency. Accompanying this inactivation is a spectroscopic change, which shifts the absorbance maximum to 392 nm. Spectral changes associated with the 1-LSD1 complex and reactivity to decreased pH and sodium borohydride treatment were suggestive of a structure involving a flavin-linked inhibitor conjugate between N5 of the flavin and the terminal carbon of the inhibitor. Using a 13C-labeled inhibitor, NMR analysis of the 1-flavin conjugate was consistent with this structural assignment. Kinetic analysis of the spectroscopic shift induced by 1 showed that the flavin adduct formed in a reaction with kinetic constants similar to those of the LSD1 inactivation process. Taken together, these data support a mechanism of LSD1 inactivation by 1 involving amine oxidation followed by Michael addition to the propargylic imine. We further examined the potential for a biotinylated analogue of 1 (1-Btn) to be used as a tool in affinity pulldown experiments. Using 1-Btn, it was feasible to selectively pull down spiked and endogenous LSD1 from HeLa cell nuclear extracts, setting the stage for activity-based demethylase proteomics.
- Subjects :
- Amino Acid Sequence
Biotinylation
Enzyme Inhibitors chemistry
Flavins pharmacology
Glutathione metabolism
HeLa Cells
Histone Demethylases
Humans
Kinetics
Magnetic Resonance Spectroscopy
Molecular Sequence Data
Oxidation-Reduction
Oxidoreductases, N-Demethylating isolation & purification
Spectrophotometry
Enzyme Inhibitors pharmacology
Oxidoreductases, N-Demethylating antagonists & inhibitors
Oxidoreductases, N-Demethylating chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 46
- Issue :
- 23
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17511474
- Full Text :
- https://doi.org/10.1021/bi700414b