Selected article for: "amino acid and catalytic mechanism"
Author: Chen, Shuai; Chen, Li-li; Luo, Hai-bin; Sun, Tao; Chen, Jing; Ye, Fei; Cai, Jian-hua; Shen, Jing-kang; Shen, Xu; Jiang, Hua-liang
Title: Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique
  • Cord-id: 35u01je6
  • Document date: 2005_1_25
    • ID: 35u01je6
    Snippet: 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
    Document: 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.

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