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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.
AuthorsKeqiang Fan, Ping Wei, Qian Feng, Sidi Chen, Changkang Huang, Liang Ma, Bing Lai, Jianfeng Pei, Ying Liu, Jianguo Chen, Luhua Lai
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 3 Pg. 1637-42 (Jan 16 2004) ISSN: 0021-9258 [Print] United States
PMID14561748 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Recombinant Proteins
  • Viral Proteins
  • Endopeptidases
  • 3C-like protease, SARS coronavirus
  • Cysteine Endopeptidases
Topics
  • Catalysis
  • Cysteine Endopeptidases
  • Endopeptidases (chemistry, isolation & purification, metabolism)
  • Escherichia coli (genetics)
  • Protein Structure, Secondary
  • Recombinant Proteins (isolation & purification, metabolism)
  • Substrate Specificity
  • Viral Proteins (chemistry, isolation & purification, metabolism)

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