FIGURE 3.

Inhibition of TGEV PLpro deubiquitinating activity by myricetin. (A) SDS-PAGE analysis of E. coli BL21 cells transformed with pET-32a-PLpro. Lane 1 represents uninduced cells while lane 2 represents induced cells. (B) Western blot of the purified TGEV PLspro protein using anti-His tag antibody. (C) AMC standard curve. The fluorescence intensity (Y) had a linear relationship with the AMC content (X) (Y = 5.8328X). (D) Double reciprocal plot for the detection of TGEV PLpro activity. The initial enzyme velocity (V) was inversely proportional to the substrate concentration ([S]) according to the Michaelis-Menten equation. The slope and intercept of the plot were utilized to compute the enzyme-substrate affinity (Km) and the maximum enzyme reaction velocity (Vmax). (E) Inhibition rate of myricetin at different concentrations on PLpro deubiquitinating enzyme activity. Myricetin inhibited TGEV PLpro activity in a dose-dependent manner, with an IC₅₀ of 6.563 μM. (F) Double reciprocal plot for the inhibition of TGEV PLpro by different concentrations of myricetin. Myricetin increased the Km of TGEV PLpro without affecting the Vmax, indicating a competitive inhibition mode.