Figure 6
Kinetics of cleavage of supercoiled pBS DNA by Ed EN. (A) Steady-state kinetics. Assays were carried out at 37°C with 2 nM Ed EN in a reaction mixture containing increasing concentrations (2-75 nM) of pBS DNA. Aliquots were withdrawn at different time-points (0-8 min) during the reaction and assayed by electrophoresis through 0.8% agarose. The concentration of the supercoiled form of pBS DNA at each time-point was quantified as described in the Materials and Methods. The disappearance of the supercoiled form of pBS DNA with time was plotted for the indicated concentration of substrate (nM) and the slopes thus obtained were taken as initial velocity at corresponding substrate concentrations. (B) and (C) DNA cleavage as a function of substrate concentration. Initial reaction velocities, obtained as described above, were plotted as a function of substrate concentration. A Lineweaver-Burk plot (C) was used to calculate the kinetic parameters Km and kcat. The data are expressed as the average of three independent determinations, as mentioned in the Results and Discussion, and the standard deviation is indicated as error bars (± SD).