Modulation of cyclin-dependent kinase 4 by binding of magnesium (II) and manganese (II) Original Research Article

All kinases require an essential divalent metal for their activity. In this study, we investigated the metal dependence of cyclin-dependent kinase 4 (CDK4). With Mg2+ as the essential metal and MgATP being the variable substrate, the maximum velocity, V, was not affected by changes in metal concentration, whereas V/K was perturbed, indicating that the metal effects were mainly derived from a change in the Km for MgATP. Analysis of the metal dependence of initial rates according to a simple metal binding model indicated the presence on enzyme of one activating metal-binding site with a dissociation constant, Kd(a), of 5±1 mM, and three inhibitory metal-binding sites with an averaged dissociation constant, Kd(i), of 12±1 mM and that the binding of metal to the activating and inhibitory sites appeared to be ordered with binding of metal to the activating site first. Substitution of Mn2+ for Mg2+ yielded similar metal dependence kinetics with a value of 1.0±0.1 and 4.7±0.1 for Kd(a) and Kd(i), respectively. The inhibition constants for the inhibition of CDK4 by MgADP and a small molecule inhibitor were also perturbed by Mg2+. Kd(a) values estimated from the metal variation of the inhibition of CDK4 by MgADP (6±3 mM) and a small molecule inhibitor (3±1 mM), were in good agreement with the Kd(a) value (5±1 mM) obtained from the metal variation of the initial rate of CDK4. By using the vanʹt Hoff plot, the temperature dependence of Kd(a) and Kd(i) yielded an enthalpy of −6.0±1.1 kcal/mol for binding of Mg2+ to the activating site and −3.2±0.6 kcal/mol for Mg2+ binding to the inhibitory sites. The values of associated entropy were also negative, indicating that these metal binding reactions were entirely enthalpy-driven. These data were consistent with metal binding to multiple sites on CDK4 that perturbs the enzyme structure, modulates the enzyme activity, and alters the affinities of inhibitor for the metal-bound enzyme species. However, the affinities of small molecule inhibitors for CDK4 were not affected by the change of metal from Mg2+ to Mn2+, suggesting that the structures of enzyme–Mg2+ and enzyme–Mn2+ were similar.