congresos y reuniones científicas
The catalytic cycle of ATP hydrolysis by dengue virus NS3 helicase in single turnover conditions
Congreso; Helicases and NTP-driven nucleic acid motors: structure, functions, mechanism and role in human disease. Faseb Summer Research Conference.; 2011
<!-- @page { margin: 2cm } P { margin-bottom: 0.21cm } --> 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 because they are the energy source of the duplex RNA unwinding by NS3. Thus, we propose to study the kinetic mechanism of nucleotide hydrolysis during the catalytic cycle of NS3. Hence, 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 carried-out at different concentrations of ATP and NS3 but, in all cases, with excess of enzyme on substrate and pseudo-first order conditions. The time courses of Pi release in the present experimental conditions were well described by mono-exponential functions of the time with apparent rate constants depending on the NS3 concentration but independent of the concentration of substrate (ATP). The absence of a lag-phase in the time course of Pi release, as well as the hyperbolic function that described the apparent rate constants as a function of [NS3] are in agreement with the consideration that the collisional complex between enzyme and substrate is produced in rapid equilibrium conditions. The minimal kinetic pathway proposed here ­-a three-step reaction model- for binding and hydrolysis of ATP by NS3 describes well the experimental results. Additionally, the global fit of the parameters of the minimal pathway to the experimental results show that K1=1709 ± 147 nM; k2=0.136 ± 0.004 s-1, k-2=0.041 ± 0.002 s-1 and K3= 0.0021 ± 0.0002 nM2 are the parameters well constrained by the data. 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 and 3% glycerol.