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PDBsum entry 1qsl
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Transferase/DNA
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PDB id
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1qsl
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' Exonucleolytic active site of the klenow fragment.
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Authors
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C.A.Brautigam,
K.Aschheim,
T.A.Steitz.
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Ref.
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Chem Biol, 1999,
6,
901-908.
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PubMed id
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Abstract
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BACKGROUND: Biochemical and biophysical experiments have shown that two
catalytically essential divalent metal ions (termed 'A' and 'B') bind to the
3'-5' exonuclease active site of the Klenow fragment (KF) of Escherichia coli
DNA polymerase I. X-ray crystallographic studies have established the normal
positions in the KF 3'-5' exonuclease (KF exo) active site of the two cations
and the single-stranded DNA substrate. Lanthanide (III) luminescence studies
have demonstrated, however, that only a single europium (III) ion (Eu3+) binds
to the KF exo active site. Furthermore, Eu3+ does not support catalysis by KF
exo or several other two-metal-ion phosphoryl-transfer enzymes. RESULTS: A
crystal structure of KF complexed with both Eu3+ and substrate single-stranded
oligodeoxynucleotide shows that a lone Eu3+ is bound near to metal-ion site A.
Comparison of this structure to a relevant native structure reveals that the
bound Eu3+ causes a number of changes to the KF exo active site. The scissile
phosphate of the substrate is displaced from its normal position by about 1 A
when Eu3+ is bound and the presence of Eu3+ in the active site precludes the
binding of the essential metal ion B. CONCLUSIONS: The substantial,
lanthanide-induced differences in metal-ion and substrate binding to KF exo
account for the inhibition of this enzyme by Eu3+. These changes also explain
the inability of KF exo to bind more than one cation in the presence of
lanthanides. The mechanistic similarity between KF exo and other two-metal-ion
phosphoryl-transfer enzymes suggests that the principles of lanthanide (III) ion
binding and inhibition ascertained from this study will probably apply to most
members of this class of enzymes.
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Secondary reference #1
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Title
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Structural principles for the inhibition of the 3'-5' Exonuclease activity of escherichia coli DNA polymerase i by phosphorothioates.
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Authors
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C.A.Brautigam,
T.A.Steitz.
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Ref.
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J Mol Biol, 1998,
277,
363-377.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. the configurations of the oxygen (or sulfur)
atoms about the scissile phosphate of normal or phos-
phorothioate DNA. The phosphates are shown in the
orientation that will occur in all other Figures. (a) Nor-
mal, or all-oxygen phosphate. The pro-R and pro-S pos-
itions are marked. The negative charge is distributed
between the non-bridging oxygens. (b) Rp phosphor-
othioate phosphate. Note that the sulfur atom has only
a single covalent bond to the phosphorus atom and is
negatively charged. The pro-S oxygen features a double
bond to phosphorus. (c) The Sp phosphorothioate.
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Figure 5.
Figure 5. Schematic drawing of the
R isomer structure. The same color-
ing scheme as in Figure 4 is used,
with the pro-R sulfur and ``attack''
water highlighted in yellow and
purple, respectively. The two Zn
ions are about 4.0 Å apart.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #2
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Title
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Structural basis for the 3'-5' Exonuclease activity of escherichia coli DNA polymerase i: a two metal ion mechanism.
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Authors
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L.S.Beese,
T.A.Steitz.
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Ref.
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Embo J, 1991,
10,
25-33.
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PubMed id
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