Publication: Steady-state cleavage kinetics for dengue virus type 2 NS2B-NS3(pro) serine protease with synthetic peptides
Issued Date
2003-02-08
Resource Type
ISSN
09298665
Other identifier(s)
2-s2.0-0037252890
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Mahidol University
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SCOPUS
Bibliographic Citation
Protein and Peptide Letters. Vol.10, No.1 (2003), 19-26
Suggested Citation
Rabuesak Khumthong, Pornwarat Niyomrattanakit, Santad Chanprapaph, Chanan Angsuthanasombat, Sakol Panyim, Gerd Katzenmeier Steady-state cleavage kinetics for dengue virus type 2 NS2B-NS3(pro) serine protease with synthetic peptides. Protein and Peptide Letters. Vol.10, No.1 (2003), 19-26. doi:10.2174/0929866033408228 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/20763
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Title
Steady-state cleavage kinetics for dengue virus type 2 NS2B-NS3(pro) serine protease with synthetic peptides
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Abstract
The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for Km, kcat and kcat/Km were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.