Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: Implications for the catalytic mechanism

Peter T. Beernink, Brent W. Segelke, Masood Z. Hadi, Jan P. Erzberger, David M. Wilson, Bernhard Rupp

Research output: Contribution to journalArticle

142 Scopus citations

Abstract

The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3′ replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 Å apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca2+ exhibits complex stimulatory and inhibitory effects on the Mg2+-dependent catalysis of Ape1, even though Ca2+ itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.

Original languageEnglish (US)
Pages (from-to)1023-1034
Number of pages12
JournalJournal of Molecular Biology
Volume307
Issue number4
DOIs
StatePublished - Apr 6 2001

Keywords

  • Abasic DNA
  • Ape1 endonuclease
  • Base excision repair
  • Enzyme mechanism
  • Metalloenzyme

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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