Pherastar optima

The measurements were carried out in a black 96-well half-area microtiter plate (Greiner) at 30°C in assay buffer (see above). The respective ligand was diluted in Master Mix (see above) and binding kinetics were initiated by the addition of 10 nmol/L KDAC8. The release of fluorogenic AMC substrate was observed in a microplate reader (PHERAstar Optima, BMG Labtech) at 450 nm (Ex: 350 nm). All solutions were pre-incubated at 30°C. Slopes were calculated for the early-stage (1,000 s to 2,000 s) and the late-stage (5,000 s to 6,000 s) using linear regression. The relative KDAC8 activity in the presence of different inhibitor concentrations was calculated as the ratio of the late-stage and early-stage slopes and normalized to free KDAC8 enzyme.

The measurements were carried out in a black 96-well microtiter plate (Greiner) at 30°C in assay buffer (see above). A serial two-fold dilution ranging from 50 µmol/L substrate, Boc-Lys(TFA)-AMC (Bachem), to 780 nmol/L, was prepared in an assay buffer containing 0.1 mg/mL trypsin. The enzymatic reaction was initiated by the addition of 10 nmol/L KDAC8. The release of fluorogenic AMC was observed in a micro plate reader (PHERAstar Optima, BMG Labtech) at 450 nm (Ex: 350 nm). The data points were plotted against time and blank corrected. For the determination of Michaelis-Menten parameters, a linear regression analysis of the initial slope (v_i) of each curve was performed. Slopes were transformed into rates of AMC product formation using a calibration curve with different AMC concentrations. The obtained enzyme conversion rates were plotted against the respective substrate concentration and fitted to the Michaelis-Menten function using GraphPad Prism software. The turnover number (k_cat) was calculated as
where v_i is the substrate conversion rate and E is the enzyme concentration. All solutions were pre-incubated at 30°C.