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PDBsum entry 1jsv
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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The cyclin-Dependent kinases cdk2 and cdk5 act by a random, Anticooperative kinetic mechanism.
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Authors
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P.M.Clare,
R.A.Poorman,
L.C.Kelley,
K.D.Watenpaugh,
C.A.Bannow,
K.L.Leach.
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Ref.
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J Biol Chem, 2001,
276,
48292-48299.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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).
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2001,
276,
48292-48299)
copyright 2001.
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