TAILIEUCHUNG - Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3

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. | Salaemae et al. Journal of Biomedical Science 2010 17 68 http content 17 1 68 The cost of publication in Journal of Biomedical Science Is borne by the National Science Council Taiwan JOURNAL OF BIOMEDICAL SCIENCE RESEARCH Open Access Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3 Wanisa Salaemae1 Muhammad Junaid1 2 Chanan Angsuthanasombat1 Gerd Katzenmeier1 Abstract 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 .

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