Mechanistic Analysis of a Suicide Inactivator of Histone Demethylase LSD1 †

Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, Maryland, United States
Biochemistry (Impact Factor: 3.02). 07/2007; 46(23):6892-902. DOI: 10.1021/bi700414b
Source: PubMed


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.

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    • "The spectral properties of adducts deduced from these two studies seem to suggest the presence of two conjugated double bonds in the propargylamine moiety of the most likely inhibition product. In a more recent work, Cole and co-workers performed experiments on a related histone demethylase flavoenzyme (Szewczuk et al. 2007) and obtained UV/VIS spectral shifts and 13 Clabelled inhibitor NMR data also suggestive of a structure involving a flavin-linked inhibitor conjugate. Nevertheless, conjugated adduct could easily be formed by post-modification of the initial complex 1 2 –FAD through acid–base proton transfers, and we feel that until a precise and complete mechanism of the MAO inhibition reaction is deduced, one cannot unequivocally assume the structure of the inhibited enzyme on the basis of both UV/VIS product characterization and the NMR structure of photoreaction product. "
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