Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique¹

Aim: To characterize enzymatic activity of severe acute respiratory syndrome
(SARS) coronavirus (CoV) 3C-like protease (3CL^pro) and its four site-directed
mutants.
Methods: Based on the fluorescence resonance energy transfer (FRET)
principle using 5-[(2^´-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and
4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer
pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV
3CL^pro proteolytic activity.
Results: The kinetic parameters of the fluorogenic
substrate have been determined as K_m=404 μmol•L^-1, k_cat=1.08 min^-1, and k_cat/K_m=2.7
mmol^-1•L•min^-1. SARS-CoV 3CL^pro showed substantial pH and temperature-triggered
activity switches, and site-directed mutagenesis analysis of SARS-CoV
3CLpro revealed that substitutions of His⁴¹, Cys¹⁴⁵, and His¹⁶³ resulted in complete
loss of enzymatic activity, while replacement of Met¹⁶² with Ala caused strongly
increased activity.
Conclusion: This present work has provided valuable information
for understanding the catalytic mechanism of SARS-CoV 3CLpro. This
FRET-based assay might supply an ideal approach for the exploration SARSCoV
3CLpro putative inhibitors.