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

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 Mg(2+) 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 K(m) 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, K(d(a)), of 5 +/-1 mM, and three inhibitory metal-binding sites with an averaged dissociation constant, K(d(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 Mn(2+) for Mg(2+) yielded similar metal dependence kinetics with a value of 1.0+/-0.1 and 4.7+/-0.1 for K(d(a)) and K(d(i)), respectively. The inhibition constants for the inhibition of CDK4 by MgADP and a small molecule inhibitor were also perturbed by Mg(2+). K(d(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 K(d(a)) value (5+/-1 mM) obtained from the metal variation of the initial rate of CDK4. By using the van't Hoff plot, the temperature dependence of K(d(a)) and K(d(i)) yielded an enthalpy of -6.0 +/- 1.1 kcal/mol for binding of Mg(2+) to the activating site and -3.2 +/- 0.6 kcal/mol for Mg(2+) 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 Mg(2+) to Mn(2+), suggesting that the structures of enzyme-Mg(2+) and enzyme-Mn(2+) were similar.