Enzymatic Kinetics of α-Glucosidase

pNPG with a concentration range of 100–4,000 μM and α-glucosidase were incubated with different concentrations of inhibitor for 10 min, respectively. 20 μl of 1.0 U/ml enzyme solutions were first mixed with 10 μl of different concentrations of inhibitors, then 50 μl PBS solutions were added, and the mixed solutions were incubated at 37°C for 10 min. Subsequently, 20 μl of pNPG solutions (1.25, 2.5, 5, 10 and 20 mM) were added, and the mixed solutions were further incubated at 37°C for 25 min, the absorbance of reaction solution was measured at 405 nm every 3 min. The kinetic parameters, Michaelis–Menten (K_m) and maximum velocity (V_max), were found using Lineweaver–Burk plots to check the mode of α-glucosidase inhibition for compounds 1 and 4. The dissociation constants between inhibitor and enzyme (K_i) were calculated from Dixon plots. Two inhibition constants, K_I or K_IS, for inhibitor binding with either free or enzyme-substrate complex, were calculated from secondary plots of the slopes of the straight lines (V_max/K_m) or vertical intercept (1/V_max) verse the concentration of inhibitors, respectively (Jenis et al., 2019).