1jsv: Difference between revisions

Revision as of 14:26, 21 February 2008

The structure of cyclin-dependent kinase 2 (CDK2) in complex with 4-[(6-amino-4-pyrimidinyl)amino]benzenesulfonamide

OverviewOverview

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.

About this StructureAbout this Structure

1JSV is a Single protein structure of sequence from Homo sapiens with as ligand. Full crystallographic information is available from OCA.

ReferenceReference

The cyclin-dependent kinases cdk2 and cdk5 act by a random, anticooperative kinetic mechanism., Clare PM, Poorman RA, Kelley LC, Watenpaugh KD, Bannow CA, Leach KL, J Biol Chem. 2001 Dec 21;276(51):48292-9. Epub 2001 Oct 16. PMID:11604388

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-[[Image:1jsv.gif|left|200px]]<br />
+[[Image:1jsv.gif|left|200px]]<br /><applet load="1jsv" size="350" color="white" frame="true" align="right" spinBox="true"
-<applet load="1jsv" size="450" color="white" frame="true" align="right" spinBox="true"
 caption="1jsv, resolution 1.96&Aring;" />
 '''The structure of cyclin-dependent kinase 2 (CDK2) in complex with 4-[(6-amino-4-pyrimidinyl)amino]benzenesulfonamide'''<br />
 ==Overview==
-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.
+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.
 ==About this Structure==
-JSV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with U55 as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1JSV OCA].
+JSV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=U55:'>U55</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1JSV OCA].
 ==Reference==
@@ Line 14: / Line 13: @@
 [[Category: Homo sapiens]]
 [[Category: Single protein]]
-[[Category: Kelley, L.C.]]
+[[Category: Kelley, L C.]]
-[[Category: Watenpaugh, K.D.]]
+[[Category: Watenpaugh, K D.]]
 [[Category: U55]]
 [[Category: atp-binding]]
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 [[Category: serine/threonine protein kinase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 12 17:44:06 2007''
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