Figure 7
Determination of CDK12 kinetic parameters.
(a) Full length CDK12/CycK1 activity was measured against varying concentrations of ATP over 3 time points (15 min, 30 min and 60 min). Velocity and Lineweaver-Burke plots are shown (left and right panels, respectively). KmATP was determined to be 2 μM +/− 0.2 μM ATP (S.D.) based on two independent experiments using three different preparations of the full length enzyme. (b) Equivalent ATP titrations and plots for the CDK12715–1052/CycK11–267 complex. KmATP was determined to be 25.5 μM +/− 0.01 μM ATP (S.D.) based on two independent experiments. (c) Full length CDK12/CycK1 activity was measured against varying concentrations of GST-CTD substrate using 10 μM ATP and a reaction time of 15 min. Experiments were performed in triplicate (velocity plot, left panel) and the average values plotted in a Lineweaver-Burke plot (right panel). KmCTD was determined to be 0.3 μM +/− 0.06 μM (S.D.). (d) Equivalent GST-CTD titrations for the CDK12715–1052/CycK11–267 complex. KmCTD was determined to be 2 μM +/− 0.7 μM (S.D.). Differences in the Km values between the full length and truncated CDK12 complexes suggest that other domains within the full length protein may contribute to substrate binding and turnover.
