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1ksp
From Proteopedia
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|PDB= 1ksp |SIZE=350|CAPTION= <scene name='initialview01'>1ksp</scene>, resolution 2.300Å | |PDB= 1ksp |SIZE=350|CAPTION= <scene name='initialview01'>1ksp</scene>, resolution 2.300Å | ||
|SITE= | |SITE= | ||
| - | |LIGAND= <scene name='pdbligand=ZN:ZINC ION'>ZN</scene> | + | |LIGAND= <scene name='pdbligand=DT:THYMIDINE-5'-MONOPHOSPHATE'>DT</scene>, <scene name='pdbligand=PST:THYMIDINE-5'-THIOPHOSPHATE'>PST</scene>, <scene name='pdbligand=ZN:ZINC+ION'>ZN</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] | + | |ACTIVITY= <span class='plainlinks'>[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] </span> |
|GENE= | |GENE= | ||
| + | |DOMAIN= | ||
| + | |RELATEDENTRY= | ||
| + | |RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1ksp FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ksp OCA], [http://www.ebi.ac.uk/pdbsum/1ksp PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=1ksp RCSB]</span> | ||
}} | }} | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: C A.Brautigam,T A.Steitz]] | [[Category: C A.Brautigam,T A.Steitz]] | ||
| - | [[Category: ZN]] | ||
[[Category: complex (dna-binding protein/dna)]] | [[Category: complex (dna-binding protein/dna)]] | ||
[[Category: exonuclease]] | [[Category: exonuclease]] | ||
[[Category: phosphorothioate]] | [[Category: phosphorothioate]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Mar 30 21:53:02 2008'' |
Revision as of 18:53, 30 March 2008
| |||||||
| , resolution 2.300Å | |||||||
|---|---|---|---|---|---|---|---|
| Ligands: | , , | ||||||
| Activity: | DNA-directed DNA polymerase, with EC number 2.7.7.7 | ||||||
| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||
| 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
1KSP 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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