Open AccessEnzymatic activity characterization of SARS coronavirus 3C‐like protease by fluorescence resonance energy transfer technique 1
Author(s) -
CHEN Shuai,
CHEN Lili,
LUO Haibin,
SUN Tao,
CHEN Jing,
YE Fei,
CAI Jianhua,
SHEN Jingkang,
SHEN Xu,
JIANG Hualiang
Publication year - 2005
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2005.00010.x
Subject(s) - protease , enzyme , förster resonance energy transfer , coronavirus , virology , covid-19 , severe acute respiratory syndrome coronavirus , chemistry , fluorescence , biochemistry , medicine , physics , pathology , quantum mechanics , infectious disease (medical specialty) , outbreak , disease
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.