Structural studies on phospho-CDK2/cyclin A bound to nitrate, a transition state analogue: implications for the protein kinase mechanism

Atlanta Cook, E D Lowe, E D Chrysina, V T Skamnaki, N G Oikonomakos, L N Johnson

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)7301-11
Number of pages11
JournalBiochemistry
Volume41
Issue number23
Publication statusPublished - 2002

Keywords

  • Adenosine Diphosphate
  • Adenylyl Imidodiphosphate
  • CDC2-CDC28 Kinases
  • Catalysis
  • Crystallization
  • Crystallography, X-Ray
  • Cyclin A
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases
  • Humans
  • Kinetics
  • Nitrates
  • Oligopeptides
  • Phosphorylation
  • Protein-Serine-Threonine Kinases
  • Substrate Specificity

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