FIGURE 5.
Kinetic characterization of DesKC autophosphorylation. A, temporal course of ATP consumption for wild-type DesKC (WT, ○) and DesKC mutants E342A (■), R343A (▴), and H188E (autophosphorylation inactive, ♦). H188E shows a basal ATPase activity corresponding to the intrinsic activity of the ABD. Wild-type DesKC displays a biphasic behavior, with an initially exponential phase followed by a second linear regime. Note the high ATP consumption rates of E342A and R343A, which showed no biphasic pattern, even in extended incubation times until NADH depletion (not shown in this plot). B, ATP dependence of wt DesKC autophosphorylation initial velocities. Pure DesKC (22 μm) was used. kcat was calculated considering the total concentration of monomer.