Kinetic basis for activation of CDK2/clyclin A by phosphorylation

The activation of most protein kinases requires phosphorylation at a conser ved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle central enzyme, CDK2/ cyclin A, in which catalytic activation depends on phosphorylation at Thr(1 60) in CDK2, The structural consequences of phosphorylation have been revea led by x-ray crystallographic studies on CDE2-cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinet ic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylatio n results in a 100,000-fold increase in catalytic efficiency and an approxi mate 1,000-fold increase in the overall turnover rate. The effects of phosp horylation on the individual steps in the catalytic reaction pathway were d etermined using solvent viscosometric techniques. It was found that the inc rease in catalytic power arises mainly from a 3,000-fold increase in the ra te of the phosphoryl group transfer step with a more moderate increase in s ubstrate binding affinity. In contrast, the rate of phosphoryl group transf er in the ATPase pathway was unaffected by phosphorylation, demonstrating t hat phosphorylation at Thr(160) does not serve to stabilize ATP in the ATPa se reaction. Thus, we hypothesize that the role of phosphorylation in the k inase reaction may be to specifically stabilize the peptide phosphoacceptor group.