2kfn: Difference between revisions

Revision as of 19:07, 21 February 2008

KLENOW FRAGMENT WITH BRIDGING-SULFUR SUBSTRATE AND MANGANESE

OverviewOverview

The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer. In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively. They serve as standards for comparison with other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis.

About this StructureAbout this Structure

2KFN is a Single protein structure of sequence from Escherichia coli with , and as ligands. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 Full crystallographic information is available from OCA.

ReferenceReference

Structures of normal single-stranded DNA and deoxyribo-3'-S-phosphorothiolates bound to the 3'-5' exonucleolytic active site of DNA polymerase I from Escherichia coli., Brautigam CA, Sun S, Piccirilli JA, Steitz TA, Biochemistry. 1999 Jan 12;38(2):696-704. PMID:9888810

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-[[Image:2kfn.gif|left|200px]]<br /><applet load="2kfn" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:2kfn.gif|left|200px]]<br /><applet load="2kfn" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="2kfn, resolution 2.03&Aring;" />
 '''KLENOW FRAGMENT WITH BRIDGING-SULFUR SUBSTRATE AND MANGANESE'''<br />
 ==Overview==
-The interaction of a divalent metal ion with a leaving 3' oxygen is a, central component of several proposed mechanisms of phosphoryl transfer., In support of this are recent kinetic studies showing that thiophilic, metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which, sulfur takes the place of the leaving oxygen. To examine the structural, basis of this phenomenon, we have solved four crystal structures of, single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging, position bound in conjunction with various metal ions at the 3'-5', exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I, from Escherichia coli. Two structures of normal ssDNA bound to KF in the, presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A, resolution, respectively. They serve as standards for comparison with, other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+, bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam, and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound, to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the, scissile phosphate were refined at 2.03-A resolution. Although the, bridging sulfur compounds bind in a manner very similar to that of the, normal oligonucleotides, the presence of the sulfur changes the metal ion, binding properties of the active site such that Mn2+ and Zn2+ are observed, at metal ion site B, but Mg2+ is not. It therefore appears that the, ability of the bridging sulfur compounds to exclude nonthiophilic metal, ions from metal ion site B explains the low activity of KF exonuclease on, these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am., Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the, substrate is influencing the binding of metal ion B prior to catalysis.
+The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer. In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively. They serve as standards for comparison with other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis.
 ==About this Structure==
-KFN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with ZN, MN and MG as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2KFN OCA].
+KFN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=ZN:'>ZN</scene>, <scene name='pdbligand=MN:'>MN</scene> and <scene name='pdbligand=MG:'>MG</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2KFN OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Escherichia coli]]
 [[Category: Single protein]]
-[[Category: Brautigam, C.A.]]
+[[Category: Brautigam, C A.]]
-[[Category: Piccirilli, J.A.]]
+[[Category: Piccirilli, J A.]]
-[[Category: Steitz, T.A.]]
+[[Category: Steitz, T A.]]
 [[Category: Sun, S.]]
 [[Category: MG]]
@@ Line 25: / Line 25: @@
 [[Category: transferase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 12:40:56 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:06:59 2008''