- 作者: Wanisa Salaemae; Muhammad Junaid; Chanan Angsuthanasombat; Gerd Katzenmeier
- 作者服務機構: Laboratory of Molecular Virology, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, Thailand
- 中文摘要: --
- 英文摘要:
Background: The dengue virus two-component protease NS2B/NS3 mediates processing of the
viral polyprotein precursor and is therefore an important determinant of virus
replication. The enzyme is now intensively studied with a view to the structure-based
development of antiviral inhibitors. Although 3-dimensional structures have now been
elucidated for a number of flaviviral proteases, enzyme-substrate interactions are
characterized only to a limited extend. The high selectivity of the dengue virus
protease for the polyprotein precursor offers the distinct advantage of designing
inhibitors with exquisite specificity for the viral enzyme. To identify important
determinants of substrate binding and catalysis in the active site of the dengue virus
NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and
I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine
substitution mutagenesis and effects on enzyme activity were fluorometrically
assayed.
Methods: Alanine substitutions were introduced by site-directed mutagenesis at residues L115,
D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins
were purified from overexpressing E. coli. Effects of these substitutions on enzymatic
activity of the NS3 protease were assayed by fluorescence release from the synthetic
model substrate GRR-amc and kinetic parameters Km, kcat and kcat/Km were
determined. Results: Kinetic data for mutant derivatives in the active site of the dengue virus NS3 protease
were essentially in agreement with a functional role of the selected residues for
substrate binding and/or catalysis. Only the L115A mutant displayed activity
comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to
alanine completely abrogated enzyme activity. A G133A mutant had an
approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for
this residue seemingly as part of the oxyanion binding hole.
Conclusions: Kinetic data obtained for mutants in the NS3 protease have confirmed predictions for
the conformation of the active site S1 and S2 pockets based on earlier observations.
The data presented herein will be useful to further explore structure-activity
relationships of the flaviviral proteases important for the structure-guided design of
novel antiviral therapeutics. - 中文關鍵字: --
- 英文關鍵字: --