CONICET | Buscador de Institutos y Recursos Humanos

The NS3 dengue virus helicase is a molecular motor protein that unwinds duplex RNA during the replication of the viral genome. The binding and hydrolysis of adenosine triphosphate (ATP) are essential for helicase function. However, the reaction mechanism that explains the coupling between nucleotide hydrolysis and duplex RNA unwinding is still unknown. Thus, we propose to study the kinetic mechanism of nucleotide hydrolysis during the catalytic cycle of NS3. According to this, we performed pre-steady state experiments of ATP hydrolysis by following orthophosphate(Pi) release in single turnover conditions. The time courses of Pi release were obtained at different concentrations of ATP and NS3 but, in all cases, with excess of enzyme on substrate and pseudo-first order conditions. When the enzyme concentration was 1.022 µM and [ATP] = 0.2, 0.051 and 0.00019 µM the time courses of Pi release were monoexponential curves with apparent rate contants: 0.048, 0.048 and 0.053 s-1 respectively. When the enzyme concentration was decreased to 0.050 and 0.014 µM with [ATP] = 0.00019 and 0.00016 µM also monoexponential curves were obtained with apparent rates constants: 0.0054 and 0.0019 s-1 respectively. These results suggests that, in the present experimental conditions the rate limiting step of ATP hydrolysis is the formation of collisional complex NS3-ATP with an apparent second order rate constant of 0.0466 µM-1 s‑1. NS3 protein was cloned from the dengue virus 2 16681 infectious clone. The recombinant protein was purified by histidine‑tag affinity chromatography. All time courses were carried-out at 25 °C in media containing: 100 mM KCl; 25 mM MOPS/K (pH=6.5); 2 mM MgCl2; 0.5 mM EDTA; 3% glycerol. With grant B412 UBACyT.