1ksp: Difference between revisions
New page: left|200px<br /><applet load="1ksp" size="450" color="white" frame="true" align="right" spinBox="true" caption="1ksp, resolution 2.300Å" /> '''DNA POLYMERASE I KL... |
No edit summary |
||
| Line 1: | Line 1: | ||
[[Image:1ksp.gif|left|200px]]<br /><applet load="1ksp" size=" | [[Image:1ksp.gif|left|200px]]<br /><applet load="1ksp" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1ksp, resolution 2.300Å" /> | caption="1ksp, resolution 2.300Å" /> | ||
'''DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX'''<br /> | '''DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX'''<br /> | ||
==Overview== | ==Overview== | ||
A two-metal-ion catalytic mechanism has previously been proposed for | A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments. | ||
==About this Structure== | ==About this Structure== | ||
1KSP 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 as [http://en.wikipedia.org/wiki/ligand ligand]. 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:// | 1KSP 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> as [http://en.wikipedia.org/wiki/ligand ligand]. 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=1KSP OCA]. | ||
==Reference== | ==Reference== | ||
| Line 14: | Line 14: | ||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: C | [[Category: C A.Brautigam,T A.Steitz]] | ||
[[Category: ZN]] | [[Category: ZN]] | ||
[[Category: complex (dna-binding protein/dna)]] | [[Category: complex (dna-binding protein/dna)]] | ||
| Line 20: | Line 20: | ||
[[Category: phosphorothioate]] | [[Category: phosphorothioate]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:37:29 2008'' | ||
Revision as of 14:37, 21 February 2008
|
DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX
OverviewOverview
A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.
About this StructureAbout this Structure
1KSP is a Single protein structure of sequence from Escherichia coli with as ligand. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 Full crystallographic information is available from OCA.
ReferenceReference
Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates., Brautigam CA, Steitz TA, J Mol Biol. 1998 Mar 27;277(2):363-77. PMID:9514742
Page seeded by OCA on Thu Feb 21 13:37:29 2008