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1krp
From Proteopedia
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(New page: 200px<br /><applet load="1krp" size="450" color="white" frame="true" align="right" spinBox="true" caption="1krp, resolution 2.200Å" /> '''DNA POLYMERASE I KL...)
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Revision as of 17:38, 20 November 2007
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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
1KRP is a Single protein structure of sequence from Escherichia coli with ZN as ligand. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 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
Page seeded by OCA on Tue Nov 20 19:45:42 2007
