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

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46 Scopus citations

Abstract

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
Pages (from-to)47-57
Number of pages11
JournalJournal of Molecular Biology
Volume287
Issue number1
DOIs
StatePublished - Mar 19 1999

Bibliographical note

Funding Information:
This work was supported by the U.S. Public Health Science grants CA53791 and ES08457 (to S.M.); by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run (to M.W.); also supported by NIH grant GM-45579, Robert A. Welch Foundation grants H-0013 and H-1238 (to J.C.L.) and by NIEHS Center grant ES06676. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Keywords

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

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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