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1ksp

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[[Image:1ksp.gif|left|200px]]
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{{STRUCTURE_1ksp| PDB=1ksp | SCENE= }}
{{STRUCTURE_1ksp| PDB=1ksp | SCENE= }}
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'''DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX'''
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===DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX===
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==Overview==
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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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(as it appears on PubMed at http://www.pubmed.gov), where 9514742 is the PubMed ID number.
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{{ABSTRACT_PUBMED_9514742}}
==About this Structure==
==About this Structure==
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[[Category: Exonuclease]]
[[Category: Exonuclease]]
[[Category: Phosphorothioate]]
[[Category: Phosphorothioate]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 23:07:34 2008''
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jul 2 10:55:00 2008''

Revision as of 07:55, 2 July 2008

Image:1ksp.png

Template:STRUCTURE 1ksp

DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX

Template:ABSTRACT PUBMED 9514742

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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