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Updated: Jul 26, 2026
Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Published on: January 27, 2012
Structural studies on phospho-CDK2/cyclin A bound to nitrate, a transition state analogue: implications for the
A Cook1, E D Lowe, E D Chrysina
1Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Rex Richards Building, South Parks Road, Oxford OX1 3QU, U.K.
Abstract:
Eukaryotic protein kinases catalyze the phosphoryl transfer of the gamma-phosphate of ATP to the serine, threonine, or tyrosine residue of protein substrates. The catalytic mechanism of phospho-CDK2/cyclin A (pCDK2/cyclin A) has been probed with structural and kinetic studies using the trigonal NO(3)(-) ion, which can be viewed as a mimic of the metaphosphate transition state. The crystal structure of pCDK2/cyclin A in complex with Mg(2+)ADP, nitrate, and a heptapeptide substrate has been determined at 2.7 A. The nitrate ion is located between the beta-phosphate of ADP and the hydroxyl group of the serine residue of the substrate. In one molecule of the asymmetric unit, the nitrate is close to the beta-phosphate of ADP (distance from the nitrate nitrogen to the nearest beta-phosphate oxygen of 2.5 A), while in the other subunit, the nitrate is closer to the substrate serine (distance of 2.1 A). Kinetic studies demonstrate that nitrate is not an effective inhibitor of protein kinases, consistent with the structural results that show the nitrate ion makes few stabilizing interactions with CDK2 at the catalytic site. The binding of orthovanadate was also investigated as a mimic of a pentavalent phosphorane intermediate of an associative mechanism for phosphoryl transfer. No vanadate was observed bound in a 3.4 A resolution structure of pCDK2/cyclin A in the presence of Mg(2+)ADP, and vanadate did not inhibit the kinase reaction. The results support the notion that the protein kinase reaction proceeds through a mostly dissociative mechanism with a trigonal planar metaphosphate intermediate rather than an associative mechanism that involves a pentavalent phosphorane intermediate.
Insights
Structural and kinetic studies of phospho-CDK2/cyclin A suggest protein kinases use a dissociative mechanism. This involves a trigonal planar metaphosphate intermediate, not an associative mechanism with a pentavalent phosphorane intermediate.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Eukaryotic protein kinases are crucial enzymes that regulate cellular processes via phosphorylation.
- Understanding the catalytic mechanism of protein kinases is essential for drug discovery and understanding disease.
Purpose of the Study:
- To elucidate the catalytic mechanism of phospho-CDK2/cyclin A (pCDK2/cyclin A) using structural and kinetic approaches.
- To investigate the role of transition state mimics, nitrate and vanadate, in understanding phosphoryl transfer.
Main Methods:
- X-ray crystallography was employed to determine the structure of pCDK2/cyclin A in complex with Mg(2+)ADP, nitrate, and a peptide substrate.
- Kinetic assays were performed to assess the inhibitory effects of nitrate and vanadate on kinase activity.
Main Results:
- Structural analysis revealed the nitrate ion positioned between ADP and the substrate serine, with varying proximity in different subunits.
- Kinetic studies showed nitrate is not an effective inhibitor, indicating limited stabilizing interactions within the CDK2 active site.
- No bound vanadate was observed in structural studies, and vanadate did not inhibit kinase activity.
Conclusions:
- The findings support a predominantly dissociative mechanism for protein kinase catalysis.
- A trigonal planar metaphosphate intermediate is favored over a pentavalent phosphorane intermediate in the associative mechanism.
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