Figure 1
From: Product release is rate-limiting for catalytic processing by the Dengue virus protease
Mutations of NS2B-NS3pro and effect on catalytic activity.
(a) Crystal structure of DENV3 NS2BH-NS3pro in complex with inhibitor Bz-Nle-KRR-H (PDB 3U1I)17. The NS3 protease is shown in grey, with the His51-Asp75-Ser135 catalytic triad in blue. NS2BH is shown in yellow with mutated residues shown either in orange (hydrophobic Leu74, Ile76 and Ile86) or green (surface exposed Glu80 and Ser83). Glu80 and Ser83 have been modelled from the native Asp80 and Thr83 present in DENV3 to mimic the DENV2 protein. (b) Effect of mutations in NS2BH on the catalytic activity of NS3pro in both glycine-linked and unlinked systems. Activity was measured in 50 mM Tris.HCl, pH 8.5, 37 °C, with 2 μM enzyme (n = 3). Cleavage of the chromogenic substrate Ac-LKRR-pNa (250 μM) was measured by monitoring the change in A405 over 10 min. The activity of NS2BH mutants is illustrated relative to that of the respective wild-type forms (c) Catalytic activity of mutants and wild-type enzymes measured in 50 mM Tris.HCl, pH 8.5, 37 °C, with 2 μM enzyme (n = 3) as a function of Ac-LKRR-pNa substrate concentration. The progress of the reaction was measured by monitoring the change in A405 over 10 min. The data displayed Michaelis-Menten-type behaviour. Main graph: Glu80Ala and Ser83Ala mutants expressed in glycine-linked (solid line) and unlinked (dashed line) systems. Inset graph: Activity of wild-type proteases, reported previously21.
