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Protein & Peptide Letters

Editor-in-Chief

ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Steady-State Cleavage Kinetics for Dengue Virus Type 2 Ns2b-Ns3(Pro) Serine Protease With Synthetic Peptides

Author(s): Rabuesak Khumthong, Pornwarat Niyomrattanakit, Santad Chanprapaph, Chanan Angsuthanasombat, Sakol Panyim and Gerd Katzenmeier

Volume 10, Issue 1, 2003

Page: [19 - 26] Pages: 8

DOI: 10.2174/0929866033408228

Price: $65

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.

Keywords: dengue virus, ns2b-ns3, serine protease, peptide, substrate, assay, kinetic, cleavage kinetics


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