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, James Lee, Sankar Mitra

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

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DNA-(Apurinic or Apyrimidinic Site) Lyase
Catalytic Domain
Mutation
Mutant Proteins
Metals
Binding Sites
Protein Conformation
Conserved Sequence
Endonucleases
DNA
Enzymes
Viperidae
Catalysis
Tryptophan
Mutagenesis
Glutamic Acid
Complementary DNA
X-Rays
Escherichia coli
Proteins

Keywords

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

ASJC Scopus subject areas

  • Virology

Cite this

Izumi, T., Malecki, J., Chaudhry, M. A., Weinfeld, M., Hill, J. H., Lee, J., & Mitra, S. (1999). Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease. Journal of Molecular Biology, 287(1), 47-57. https://doi.org/10.1006/jmbi.1999.2573

Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease. / Izumi, Tadahide; Malecki, Jedrzej; Chaudhry, M. Ahmad; Weinfeld, Michael; Hill, Jeff H.; Lee, James; Mitra, Sankar.

In: Journal of Molecular Biology, Vol. 287, No. 1, 19.03.1999, p. 47-57.

Research output: Contribution to journalArticle

Izumi, T, Malecki, J, Chaudhry, MA, Weinfeld, M, Hill, JH, Lee, J & Mitra, S 1999, 'Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease', Journal of Molecular Biology, vol. 287, no. 1, pp. 47-57. https://doi.org/10.1006/jmbi.1999.2573
Izumi, Tadahide ; Malecki, Jedrzej ; Chaudhry, M. Ahmad ; Weinfeld, Michael ; Hill, Jeff H. ; Lee, James ; Mitra, Sankar. / Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease. In: Journal of Molecular Biology. 1999 ; Vol. 287, No. 1. pp. 47-57.
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