Original Article

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

Shuai CHEN, Li-li CHEN, Hai-bin LUO, Tao SUN, Jing CHEN, Fei YE, Jian-hua CAI, Jing-kang SHEN, Xu SHEN, Hua-liang JIANG


Aim: To characterize enzymatic activity of severe acute respiratory syndrome
(SARS) coronavirus (CoV) 3C-like protease (3CLpro) and its four site-directed
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
3CLpro proteolytic activity.
Results: The kinetic parameters of the fluorogenic
substrate have been determined as Km=404 μmol•L-1, kcat=1.08 min-1, and kcat/Km=2.7
mmol-1•L•min-1. SARS-CoV 3CLpro 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.