Kinetic basis for activation of CDK2/cyclin A by phosphorylation Journal Article
| Authors: | Hagopian, J. C.; Kirtley, M. P.; Stevenson, L. M.; Gergis, R. M.; Russo, A. A.; Pavletich, N. P.; Parsons, S. M.; Lew, J. |
| Article Title: | Kinetic basis for activation of CDK2/cyclin A by phosphorylation |
| Abstract: | The activation of most protein kinases requires phosphorylation at a conserved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which catalytic activation depends on phosphorylation at Thr160 in CDK2. The structural consequences of phosphorylation have been revealed by x-ray crystallographic studies on CDK2/cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinetic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylation results in a 100,000-fold increase in catalytic efficiency and an approximate 1,000-fold increase in the overall turnover rate. The effects of phosphorylation on the individual steps in the catalytic reaction pathway were determined using solvent viscosometric techniques. It was found that the increase in catalytic power arises mainly from a 3,000-fold increase in the rate of the phosphoryl group transfer step with a more moderate increase in substrate binding affinity. In contrast, the rate of phosphoryl group transfer in the ATPase pathway was unaffected by phosphorylation, demonstrating that phosphorylation at Thr160 does not serve to stabilize ATP in the ATPase reaction. Thus, we hypothesize that the role of phosphorylation in the kinase reaction may be to specifically stabilize the peptide phosphoacceptor group. |
| Keywords: | binding affinity; cell cycle; enzyme activation; enzyme activity; phosphorylation; enzyme phosphorylation; kinetics; recombinant fusion proteins; isotope labeling; protein-serine-threonine kinases; binding site; thermodynamics; threonine; catalysis; adenosine triphosphate; enzyme kinetics; cyclin-dependent kinases; cyclin a; cyclin dependent kinase; adenosine triphosphatases; viscosity; solvents; cyclic amp dependent protein kinase; cyclin-dependent kinase 2; enzyme stability; chromatography, ion exchange; phosphorus 32; cdc2-cdc28 kinases; humans; human; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 276 |
| Issue: | 1 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2001-01-05 |
| Start Page: | 275 |
| End Page: | 280 |
| Language: | English |
| DOI: | 10.1074/jbc.M007337200 |
| PUBMED: | 11029468 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 21 May 2015 -- Source: Scopus |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work