title: Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique.
abstract: 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.
authors.affiliation: 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.
authors.forename: Shuai
authors.initials: S
authors.lastname: Chen
authors.forename: Li-li
authors.initials: LL
authors.lastname: Chen
authors.forename: Hai-bin
authors.initials: HB
authors.lastname: Luo
authors.forename: Tao
authors.initials: T
authors.lastname: Sun
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Fei
authors.initials: F
authors.lastname: Ye
authors.forename: Jian-hua
authors.initials: JH
authors.lastname: Cai
authors.forename: Jing-kang
authors.initials: JK
authors.lastname: Shen
authors.forename: Xu
authors.initials: X
authors.lastname: Shen
authors.forename: Hua-liang
authors.initials: HL
authors.lastname: Jiang
mesh_terms: 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
chemical_list: D014764:Viral Proteins; D006639:Histidine; D008715:Methionine; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases; D003545:Cysteine; D000409:Alanine