TY - JOUR AU - CHEN Shuai AU - CHEN Li-li AU - LUO Hai-bin AU - SUN Tao AU - CHEN Jing AU - YE Fei AU - CAI Jian-hua AU - SHEN Jing-kang AU - SHEN Xu AU - JIANG Hua-liang PY - 2016 TI - Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique 1 JF - Acta Pharmacologica Sinica; Vol 26, No 1 (January 2005): Acta Pharmacologica Sinica Y2 - 2016 KW - N2 - 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 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 3CLpro. This FRET-based assay might supply an ideal approach for the exploration SARSCoV 3CLpro putative inhibitors. UR - http://www.chinaphar.com/article/view/3532