PDBsum entry 1jsv

Transferase

PDB id: 1jsv

Contents

Protein chain

288 a.a. *

Ligands

U55

Waters ×368

* Residue conservation analysis

PDB id:

1jsv

Name:

Transferase

Title:

The structure of cyclin-dependent kinase 2 (cdk2) in complex with 4- [(6-amino-4-pyrimidinyl)amino]benzenesulfonamide

Structure:

Cell division protein kinase 2. Chain: a. Synonym: cdk2, p33 protein kinase. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108. Expression_system_cell_line: high-five insect cells.

Resolution:

1.96Å R-factor: 0.185

Authors:

K.D.Watenpaugh,L.C.Kelley

Key ref:

P.M.Clare et al. (2001). The cyclin-dependent kinases cdk2 and cdk5 act by a random, anticooperative kinetic mechanism. J Biol Chem, 276, 48292-48299. PubMed id: 11604388 DOI: 10.1074/jbc.M102034200

Date:

19-Aug-01 Release date: 29-Aug-01

Protein chain

P24941  (CDK2_HUMAN) -  Cyclin-dependent kinase 2 from Homo sapiens

Seq: 298 a.a.

Struc: 288 a.a.

Enzyme reactions

• Enzyme class: E.C.2.7.11.22 - cyclin-dependent kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] (Bound ligand (Het Group name = U55) matches with 40.62% similarity) + ADP + H(+)

L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] (Bound ligand (Het Group name = U55) matches with 40.62% similarity) + ADP + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M102034200

J Biol Chem 276:48292-48299 (2001)

PubMed id: 11604388

The cyclin-dependent kinases cdk2 and cdk5 act by a random, anticooperative kinetic mechanism.

P.M.Clare, R.A.Poorman, L.C.Kelley, K.D.Watenpaugh, C.A.Bannow, K.L.Leach.

ABSTRACT

cdk2.cyclin E and cdk5.p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2.GST-cyclin E and cdk5.GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K(i) value of 0.6 mm. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K(i) value of 2 microm. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2.GST-cyclin E the kinetic parameters were determined to be K(m, ATP) = 3.6 +/- 1.0 microm, K(m, peptide) = 4.6 +/- 1.4 microm, and the anticooperativity factor, alpha = 130 +/- 44. For cdk5.GST-p25, the K(m, ATP) = 3.2 +/- 0.7 microm, K(m, peptide) = 1.6 +/- 0.3 microm, and alpha = 7.2 +/- 1.8.

Selected figure(s)

Figure 4.
Fig. 4. Crystal structure of PNU 112455A bound to cdk2. A, schematic tracing of the cdk2 backbone shown with ATP (brown) (35) and PNU 112455A (green). Software in Refs. 63 and 64 was used to generate the figure. B, a close-up view of the ligand and the surrounding cdk2 environment, showing hydrogen bonds (dotted lines) made by PNU 112455A (green and heteroatom colors), and the relationship of this ligand to the ATP binding site determined in Refs. 35 and 46 (orange and heteroatom colors).

Figure 5.
Fig. 5. PKV is a competitive cdk inhibitor. A, Lineweaver-Burk plot of PKV versus peptide substrate with cdk5·GST-p25. B and C, Lineweaver-Burk plots of PKV versus ATP with cdk2·GST-cyclin E (B) and cdk5·GST-p25 (C). PKV concentrations: circle , 0 mM; , 1 mM; , 2 mM; , 4 mM; 5 mM.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2001, 276, 48292-48299) copyright 2001.

Figures were selected by an automated process.