In vitro LSD1 enzyme kinetic assay for determination of kinact/KI values

Kinetics of in vitro LSD1 enzyme activity was measured with a peroxidase-coupled assay (35). Various concentrations of the compounds were incubated with 500 μM mono-methylated Lys 4 histone H3 peptide that was synthesized by Scrum Inc. in assay buffer [50 mM tris-HCl (pH 8.0) and 0.1% BSA]. After the addition of horseradish peroxidase (20 μg/ml; 31490, Thermo Fisher Scientific) and 50 μM AmplexRed reagent (A22177, Thermo Fisher Scientific), enzyme reaction was initiated by incubation with 20 nM recombinant LSD1/CoREST. The hydrogen peroxide production during amine oxidase reaction by LSD1 was monitored by AmplexRed reagent. The fluorescence signals of the resultant product resorufin were detected at 585 nm, with the excitation at 570 nm, by using Spectra Max (Molecular Devices). Because the compounds were irreversible inhibitors, their progress curve of fluorescent signals with background subtraction was fitted with Eq. 1, using GraphPad Prism 5 Software (GraphPad Software Inc.)

where v_i represents initial velocity, and k_obs and t demonstrate pseudo–first-order rate constant and time, respectively. y₀ is the product when t is 0. The estimated k_obs were replotted as a function of inhibitor concentration, and k_inact or K_I values were calculated from Eq. 2

where K_m value represents the concentration of histone H3 peptide substrate, evaluated at 20 μM.