Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique.
OBJECTIVE
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 Km=404 micromol.L(-1), kcat=1.08 min(-1), and kcat/Km=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 His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity.


CONCLUSIONS
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.
Chen
Shuai
S
Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 201203, China.
Chen
Li-li
LL
Luo
Hai-bin
HB
Sun
Tao
T
Chen
Jing
J
Ye
Fei
F
Cai
Jian-hua
JH
Shen
Jing-kang
JK
Shen
Xu
X
Jiang
Hua-liang
HL
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000086782:Coronavirus 3C Proteases; D003545:Cysteine / Q000235:genetics; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
D014764:Viral Proteins; D006639:Histidine; D008715:Methionine; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases; D003545:Cysteine; D000409:Alanine