title: 3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism.
abstract: SARS 3C-like proteinase has been proposed to be a key enzyme for drug design against SARS. Lack of a suitable assay has been a major hindrance for enzyme kinetic studies and a large-scale inhibitor screen for SARS 3CL proteinase. Since SARS 3CL proteinase belongs to the cysteine protease family (family C3 in clan CB) with a chymotrypsin fold, it is important to understand the catalytic mechanism of SARS 3CL proteinase to determine whether the proteolysis proceeds through a general base catalysis mechanism like chymotrypsin or an ion pair mechanism like papain. We have established a continuous colorimetric assay for SARS 3CL proteinase and applied it to study the enzyme catalytic mechanism. The proposed catalytic residues His41 and Cys145 were confirmed to be critical for catalysis by mutating to Ala, while the Cys145 to Ser mutation resulted in an active enzyme with a 40-fold lower activity. From the pH dependency of catalytic activity, the pK(a)'s for His41 and Cys145 in the wild-type enzyme were estimated to be 6.38 and 8.34, while the pK(a)'s for His41 and Ser145 in the C145S mutant were estimated to be 6.15 and 9.09, respectively. The C145S mutant has a normal isotope effect in D(2)O for general base catalysis, that is, reacts slower in D(2)O, while the wild-type enzyme shows an inverse isotope effect which may come from the lower activation enthalpy. The pK(a) values measured for the active site residues and the activity of the C145S mutant are consistent with a general base catalysis mechanism and cannot be explained by a thiolate-imidazolium ion pair model.
authors.affiliation: State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
authors.forename: Changkang
authors.initials: C
authors.lastname: Huang
authors.forename: Ping
authors.initials: P
authors.lastname: Wei
authors.forename: Keqiang
authors.initials: K
authors.lastname: Fan
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Luhua
authors.initials: L
authors.lastname: Lai
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D002384:Catalysis; D003124:Colorimetry / Q000379:methods; D000086782:Coronavirus 3C Proteases; D003545:Cysteine / Q000235:genetics; D003546:Cysteine Endopeptidases; D041961:Deuterium Exchange Measurement; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics; D012694:Serine / Q000235:genetics; D012997:Solvents; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D012997:Solvents; D014764:Viral Proteins; D012694:Serine; D006639:Histidine; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases; D003545:Cysteine; D000409:Alanine