Figure 3
From: Product release is rate-limiting for catalytic processing by the Dengue virus protease
Single turnover enzyme kinetics.
Hydrolysis of the para-nitroanalide substrate Ac-LKRR-pNa (25 μM) by the wild-type form of the glycine-linked DENV2 protease complex NS2BglyNS3pro. Activity measured in 50 mM Tris.HCl, pH 8.5 at 37 °C. (a) Product formation monitored by diode array between 310-725 nm. For illustration only data measured in 50 ms intervals are shown. The arrow indicates increasing product concentrations at optimal λ = 405 nm (Ɛ405 = 9,500 M−1cm−1). (b) Time course for product formation. The data were fitted to a first-order exponential (providing an estimate of kobs) using Reactlab software (eq. 3). Corresponding data for the unlinked wild-type system and the various mutants tested are shown in the Supplementary Section (see supplementary Fig. S3).
