Activity of Recombinant Dengue 2 Virus NS3 Protease in the Presence of a Truncated NS2B Co-factor, Small Peptide Substrates, and Inhibitors
MetadataShow full item record
Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mM, 1 mMCHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4′) was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.
Journal of Biological Chemistry
This research was originally published in Journal of Biological Chemistry (JBC). Leung et al, Activity of Recombinant Dengue 2 Virus NS3 Protease in the Presence of a Truncated NS2B Co-factor, Small Peptide Substrates, and Inhibitors, Journal of Biological Chemistry (JBC), Vol. 276, No. 49, pp. 45762–45771, 2001. Copyright the American Society for Biochemistry and Molecular Biology. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive version.
Medicinal and Biomolecular Chemistry not elsewhere classified