Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment

Chad A Brautigam, Kathryn Aschheim, Thomas A. Steitz

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

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 Å 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.

Original languageEnglish (US)
Pages (from-to)901-908
Number of pages8
JournalChemistry and Biology
Volume6
Issue number12
DOIs
StatePublished - Dec 1999

Fingerprint

Lanthanoid Series Elements
DNA Polymerase I
Catalytic Domain
Ions
Metal ions
Metals
Exonucleases
Substrates
Enzymes
Cations
Europium
Oligodeoxyribonucleotides
Single-Stranded DNA
Luminescence
Catalysis
Catalyst supports
Escherichia coli
Crystal structure
Phosphates
Binding Sites

Keywords

  • -3'-5' exonuclease
  • Europium (III)
  • Lanthanide (III)
  • Two-metal-ion mechanism
  • X-ray crystallography

ASJC Scopus subject areas

  • Organic Chemistry

Cite this

Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment. / Brautigam, Chad A; Aschheim, Kathryn; Steitz, Thomas A.

In: Chemistry and Biology, Vol. 6, No. 12, 12.1999, p. 901-908.

Research output: Contribution to journalArticle

@article{6fd6ebe7040649f0b52c08a20f70e279,
title = "Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment",
abstract = "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 {\AA} 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.",
keywords = "-3'-5' exonuclease, Europium (III), Lanthanide (III), Two-metal-ion mechanism, X-ray crystallography",
author = "Brautigam, {Chad A} and Kathryn Aschheim and Steitz, {Thomas A.}",
year = "1999",
month = "12",
doi = "10.1016/S1074-5521(00)80009-5",
language = "English (US)",
volume = "6",
pages = "901--908",
journal = "Cell Chemical Biology",
issn = "2451-9448",
publisher = "Elsevier Inc.",
number = "12",

}

TY - JOUR

T1 - Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment

AU - Brautigam, Chad A

AU - Aschheim, Kathryn

AU - Steitz, Thomas A.

PY - 1999/12

Y1 - 1999/12

N2 - 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 Å 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.

AB - 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 Å 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.

KW - -3'-5' exonuclease

KW - Europium (III)

KW - Lanthanide (III)

KW - Two-metal-ion mechanism

KW - X-ray crystallography

UR - http://www.scopus.com/inward/record.url?scp=0033485840&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033485840&partnerID=8YFLogxK

U2 - 10.1016/S1074-5521(00)80009-5

DO - 10.1016/S1074-5521(00)80009-5

M3 - Article

C2 - 10631518

AN - SCOPUS:0033485840

VL - 6

SP - 901

EP - 908

JO - Cell Chemical Biology

JF - Cell Chemical Biology

SN - 2451-9448

IS - 12

ER -