Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique
AbstractAim:To characterize enzymatic activity of severe acute respiratory syndrome (SARS) coronavirus (CoV) 3C-like protease (3CLpro) 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 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 SARS-CoV 3CLpro putative inhibitors.