Thermodynamic activation parameters of the ATPase activity of dengue virus NS3 helicase

LA CABABIE; SB. KAUFMAN; JJ INCICCO; T. MARQUES; RM GONZÁLEZ LEBRERO

San MIguel de Tucumán

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) IX IberoAmerican Congress of Biophysics. XLV Reunion Anual SAB 2016.; 2016

P { margin-bottom: 0.21cm; }Dengue virus NS3(non-structural protein 3) is a viral helicase that catalyzes thehydrolysis of ATP and couples the free energy of this reaction todrive its translocation along single strands and unwind doublestrands of RNA. In this work, we characterize the effect oftemperature on the enzymatic catalysis of ATP hydrolysis by the NS3helicase in the absence of RNA. We obtained thedependence of ATPase activity of NS3 on substrate concentration atdifferent temperatures ranging from 5 ºC to 30 ºC, by measuring theinitial rate of phosphate release from ATP hydrolysis. All reactionswere performed in the presence of 10 nM NS3 in media containing 25 mMMOPS; 1.5 mM free Mg2+ ; 0.50 mM EDTA; 100 mM (KCl + KOH);10 mM NaCl; 0.2 mM CHAPS; pH = 6.5 (adjusted with KOH at eachtemperature tested). ATPase activitydependence on substrate concentration was well described by simplehyperbolic functions at all temperatures tested, whose parameters,kcat and KM, were obtained by least-square non-linear regressionanalysis. We observed that while kcat and kcat/KM increasedexponentially as a function of temperature, KM remained approximatelyconstant, at a value of (0.12 ± 0.03) mM. We analyzed thetemperature dependence of kcat by means of transition state theory,employing Eyring equation, which allowed us to estimate thermodynamicactivation parameters associated to this kinetic constant. The activationenthalpy and entropy were ∆?Ho = (16 ± 2)kcal mol−1 and ∆?So = (1 ±56) 10-4 kcal K-1 mol−1respectively. Therefore, at 25 ºC, the free energy barrier, ∆?Go= (16 ± 4) kcal mol−1 , is mostly enthalpic. Comparisonwith bibliographic values of ∆?Ho and ∆?Sofor the nonenzymatic hydrolysis of ATP suggests that, in the range oftemperature tested, NS3 enhances the reaction rate mainly by reducingthe enthalpic component of the free energy barrier in ∼ 10 kcalmol−1 .