Stopped-flow measurements with fluorescence detection were carried out using a SX.20 stopped-flow spectrometer (Applied Photophysics Ltd., United Kingdom) equipped with a 150-W Xe arc lamp and an optical cell with 2 mm path length. The dead time of the instrument is 1.0 ms. For the analysis of enzyme–substrate interactions, the FRET-S substrate modified with the dye–quencher pair Edans/Dabcyl was utilized. The fluorescence of Edans was excited at λex = 340 nm and monitored at λem > 435 nm as transmitted by filter GG-435 (Schott, Mainz, Germany). The binding of Mpro to PF-00835231 was monitored by means of changes in intrinsic fluorescence intensity of the inhibitor. The excitation wavelength was 300 nm, and the emission was monitored using long-pass wavelength filters at λem > 370 nm (Corion filter LG-370).
The enzyme was placed in one of the instrument’s syringes and rapidly mixed in the reaction chamber with the substrate, inhibitor, or a substrate/inhibitor mixture from another syringe. The concentration of FRET-S in all the experiments was 2.5 μM, while concentrations of Mpro or its C145A mutant were varied from 0.1 to 3.0 μM. The reported concentrations of reactants are those in the reaction chamber after the mixing. All experiments were conducted at 25 °C in the reaction buffer.
The enzyme was placed in one of the instrument’s syringes and rapidly mixed in the reaction chamber with the substrate, inhibitor, or a substrate/inhibitor mixture from another syringe. The concentration of FRET-S in all the experiments was 2.5 μM, while concentrations of Mpro or its C145A mutant were varied from 0.1 to 3.0 μM. The reported concentrations of reactants are those in the reaction chamber after the mixing. All experiments were conducted at 25 °C in the reaction buffer.