Kinetic study of BChE inhibition

Kinetic studies were performed using the same protocol as the BChE inhibitory activity assay, and the substrate BTCI was used in a series of concentrations, 0.2, 0.4, 0.5, 0.6, and 0.7 mM. The test compound concentrations were set according to the inhibition IC₅₀ of each compound. The enzymatic reaction was extended to 15 min before the detection of the absorbance. Lineweaver–Burk plots were generated to obtain the kinetic parameters, Michaelis constant (Km) and maximum velocity (Vmax), according to which inhibition modes were determined. The value of the inhibitor constant Ki was determined based on a secondary plot of K as a function of the inhibitor concentration [I] for competitive inhibitors (Copeland, 2000).