1kfs
From Proteopedia
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| - | [[Image:1kfs.gif|left|200px]] | + | [[Image:1kfs.gif|left|200px]] |
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| - | '''DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX''' | + | {{Structure |
| + | |PDB= 1kfs |SIZE=350|CAPTION= <scene name='initialview01'>1kfs</scene>, resolution 2.100Å | ||
| + | |SITE= | ||
| + | |LIGAND= <scene name='pdbligand=ZN:ZINC+ION'>ZN</scene> and <scene name='pdbligand=MG:MAGNESIUM ION'>MG</scene> | ||
| + | |ACTIVITY= [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] | ||
| + | |GENE= | ||
| + | }} | ||
| + | |||
| + | '''DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX''' | ||
| + | |||
==Overview== | ==Overview== | ||
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==About this Structure== | ==About this Structure== | ||
| - | 1KFS is a [ | + | 1KFS is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KFS OCA]. |
==Reference== | ==Reference== | ||
| - | 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:[http:// | + | 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:[http://www.ncbi.nlm.nih.gov/pubmed/9514742 9514742] |
[[Category: DNA-directed DNA polymerase]] | [[Category: DNA-directed DNA polymerase]] | ||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
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[[Category: phosphorothioate]] | [[Category: phosphorothioate]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 12:16:51 2008'' |
Revision as of 10:16, 20 March 2008
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| , resolution 2.100Å | |||||||
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| Ligands: | and | ||||||
| Activity: | DNA-directed DNA polymerase, with EC number 2.7.7.7 | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX
Overview
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
1KFS is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
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
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