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1krp

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(New page: 200px<br /><applet load="1krp" size="450" color="white" frame="true" align="right" spinBox="true" caption="1krp, resolution 2.200&Aring;" /> '''DNA POLYMERASE I KL...)
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[[Image:1krp.gif|left|200px]]<br /><applet load="1krp" size="450" color="white" frame="true" align="right" spinBox="true"
 
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caption="1krp, resolution 2.200&Aring;" />
 
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'''DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX'''<br />
 
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==Overview==
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==DNA polymerase I Klenow fragment (E.C.2.7.7.7) mutant/DNA complex==
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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.
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<StructureSection load='1krp' size='340' side='right'caption='[[1krp]], [[Resolution|resolution]] 2.20&Aring;' scene=''>
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== Structural highlights ==
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<table><tr><td colspan='2'>[[1krp]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KRP OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1KRP FirstGlance]. <br>
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</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.2&#8491;</td></tr>
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<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=PST:THYMIDINE-5-THIOPHOSPHATE'>PST</scene>, <scene name='pdbligand=ZN:ZINC+ION'>ZN</scene></td></tr>
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<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1krp FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1krp OCA], [https://pdbe.org/1krp PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1krp RCSB], [https://www.ebi.ac.uk/pdbsum/1krp PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1krp ProSAT]</span></td></tr>
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</table>
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== Function ==
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[https://www.uniprot.org/uniprot/DPO1_ECOLI DPO1_ECOLI] In addition to polymerase activity, this DNA polymerase exhibits 3' to 5' and 5' to 3' exonuclease activity. It is able to utilize nicked circular duplex DNA as a template and can unwind the parental DNA strand from its template.
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== Evolutionary Conservation ==
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[[Image:Consurf_key_small.gif|200px|right]]
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Check<jmol>
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<jmolCheckbox>
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<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/kr/1krp_consurf.spt"</scriptWhenChecked>
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<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
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<text>to colour the structure by Evolutionary Conservation</text>
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</jmolCheckbox>
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</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1krp ConSurf].
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<div style="clear:both"></div>
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==About this Structure==
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==See Also==
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1KRP 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://ispc.weizmann.ac.il/oca-bin/ocashort?id=1KRP OCA].
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*[[DNA polymerase 3D structures|DNA polymerase 3D structures]]
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__TOC__
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==Reference==
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</StructureSection>
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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://ispc.weizmann.ac.il//pmbin/getpm?pmid=9514742 9514742]
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[[Category: DNA-directed DNA polymerase]]
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[[Category: Escherichia coli]]
[[Category: Escherichia coli]]
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[[Category: Single protein]]
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[[Category: Large Structures]]
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[[Category: Brautigam, C.A.]]
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[[Category: Brautigam CA]]
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[[Category: Steitz, T.A.]]
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[[Category: Steitz TA]]
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[[Category: ZN]]
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[[Category: complex (hydrolase/dna)]]
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[[Category: exonuclease]]
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[[Category: phosphorothioate]]
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''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 19:45:42 2007''
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Current revision

DNA polymerase I Klenow fragment (E.C.2.7.7.7) mutant/DNA complex

PDB ID 1krp

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