Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease

Tadahide Izumi, Jedrzej Malecki, M. Ahmad Chaudhry, Michael Weinfeld, Jeff H. Hill, J. Ching Lee, Sankar Mitra

Research output: Contribution to journalArticlepeer-review

42 Scopus citations


The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the K(m) of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their k(cat) values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure.

Original languageEnglish (US)
Pages (from-to)47-57
Number of pages11
JournalJournal of Molecular Biology
Issue number1
StatePublished - Mar 19 1999
Externally publishedYes


  • 3'phosphoesterase
  • AP endonuclease
  • DNA repair
  • Missense mutation
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Structural Biology


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