Figure 2.

NS3h_1b(con1) catalyzed DNA unwinding at various NTP concentrations. MBHAs were performed as described in Figure 1 except that various concentrations of NTPs were used and MgCl₂ was added to each reaction such that only 0.25 mM free Mg²⁺ was present at the start of each reaction, assuming Mg²⁺ forms a 1:1 complex with ATP with a high affinity. For example, reactions with 1 mM ATP, contained 1.25 mM total MgCl₂, whereas those with 0.2 mM dTTP contained 0.45 mM MgCl₂. (a) Comparison of reactions activated by GTP (squares), ATP (triangles), and dTTP (diamonds). The data are globally fit to a model for substrate inhibition (equation (2), solid lines), with a K_a describing NTP activation of 3.8 mM and a K_i describing NTP inhibition of 0.09 mM. In this model each NTP supports a different V_max: GTP= 0.41 nM/min, ATP = 10 nM/min, and dTTP = 50 nM/min. In an alternate analysis, data for reaction performed at or below 1 mM NTP were fit to the Michaelis-Menten equation (dotted lines) with the following constants: K_m (GTP)= 0.41 mM, V_max (GTP) = 0.05 nM/min, K_m (ATP)= 0.07 mM, V_max (ATP) = 0.79 nM/min, K_m (dTTP)= 0.30 mM, V_max (dTTP) = 5.3 nM/min. (b) Initial rates of NS3h catalyzed DNA unwinding in the presence of various concentrations of ATP and GTP. Data are fit to a model where GTP acts as a non-competitive inhibitor while ATP acts as an activator and substrate inhibitor (equation (3)) with a K_a (ATP) of 1.3 mM, K_i (ATP) of 0.69 mM, K_ii (GTP) of 0.65 mM, and a V_max of 1.6 nM/min.