title: Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase.
abstract: The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1' positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more beta-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.
authors.affiliation: State Key Laboratory of Structural Chemistry of Stable and Unstable Species, College of Chemistry, Peking University, Beijing 100871, China.
authors.forename: Keqiang
authors.initials: K
authors.lastname: Fan
authors.forename: Ping
authors.initials: P
authors.lastname: Wei
authors.forename: Qian
authors.initials: Q
authors.lastname: Feng
authors.forename: Sidi
authors.initials: S
authors.lastname: Chen
authors.forename: Changkang
authors.initials: C
authors.lastname: Huang
authors.forename: Liang
authors.initials: L
authors.lastname: Ma
authors.forename: Bing
authors.initials: B
authors.lastname: Lai
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Pei
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Jianguo
authors.initials: J
authors.lastname: Chen
authors.forename: Luhua
authors.initials: L
authors.lastname: Lai
mesh_terms: D002384:Catalysis; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases