Figure 3.

Cooperativity between nucleotide and Mg²⁺ binding to CDK2. (A) Dependence of the multiple turnover kinase reaction on the concentration of ATP at constant [Mg²⁺]_total. Histone substrate was saturating and kinase activity was measured using a coupled assay (see Experimental Methods for details). Concentration of total Mg²⁺ was fixed at either 5 or 8 mM. (B) Multiple turnover kinetics in which the concentration of ATP·Mg²⁺ was varied at a fixed concentration of either 1 (black) or 10 mM (red) free Mg²⁺. Velocity was fit by the Michaelis–Menten equation (lines). (C–E) Resulting steady state constants from these experiments and from data collected at additional concentrations of free Mg²⁺ (see Supporting Information Figures 4–14). (C) Value of k_cat/K_M^ATP·Mg was fit to a hyperbolic binding equation, indicating a very weak affinity of ∼10 mM for Mg²⁺ binding to free enzyme in the absence of nucleotide. (D) Value of k_cat shows a complex dependence on the concentration of free Mg²⁺ with a highly cooperative stimulation at very low Mg²⁺ and a modest decrease in k_cat between 1 and 20 mM Mg²⁺. Inset: log10 scale. (E) Value of K_M for ATP·Mg²⁺ is dramatically decreased by binding of a second Mg²⁺ ion. (F) Dissociation constant for ADP was determined by isothermal calorimetry as a function of total Mg²⁺ (see Supporting Information for details).