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

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 3CL(pro) revealed that substitutions of His(41), Cys(145), and His(163) resulted in complete loss of enzymatic activity, while replacement of Met(162) with Ala caused strongly increased activity. CONCLUSION: This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CL(pro). This FRET-based assay might supply an ideal approach for the exploration SARS-CoV 3CL(pro) putative inhibitors.