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

Abstract

Aim: 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, k cat=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 Met 162 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.