1kfs: Difference between revisions
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|PDB= 1kfs |SIZE=350|CAPTION= <scene name='initialview01'>1kfs</scene>, resolution 2.100Å | |PDB= 1kfs |SIZE=350|CAPTION= <scene name='initialview01'>1kfs</scene>, resolution 2.100Å | ||
|SITE= | |SITE= | ||
|LIGAND= <scene name='pdbligand= | |LIGAND= <scene name='pdbligand=DA:2'-DEOXYADENOSINE-5'-MONOPHOSPHATE'>DA</scene>, <scene name='pdbligand=DC:2'-DEOXYCYTIDINE-5'-MONOPHOSPHATE'>DC</scene>, <scene name='pdbligand=DG:2'-DEOXYGUANOSINE-5'-MONOPHOSPHATE'>DG</scene>, <scene name='pdbligand=DT:THYMIDINE-5'-MONOPHOSPHATE'>DT</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</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=1kfs FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1kfs OCA], [http://www.ebi.ac.uk/pdbsum/1kfs PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=1kfs RCSB]</span> | |||
}} | }} | ||
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[[Category: Brautigam, C A.]] | [[Category: Brautigam, C A.]] | ||
[[Category: Steitz, T A.]] | [[Category: Steitz, T A.]] | ||
[[Category: complex (hydrolase/dna)]] | [[Category: complex (hydrolase/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 21:47:58 2008'' | ||
Revision as of 21:48, 30 March 2008
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| , resolution 2.100Å | |||||||
|---|---|---|---|---|---|---|---|
| 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
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
1KFS is a Single protein structure of sequence from Escherichia coli. 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
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