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

The apurinic/apyrimidinic endonucleases (APE) contain several highly conser ved sequence motifs. The glutamic acid residue in a consensus motif, LQE96T K98 in human APE (hAPE-1), is crucial because of its role in coordinating M g2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolatio n of an intragenic suppressor with a second site mutation, K98R. Although t he K-m of the suppressor mutant was about sixfold higher than that of the w ild-type enzyme, their k(cat) values were similar for AP endonuclease activ ity. These results suggest that the E96A mutation affects only the DNA-bind ing step, but not the catalytic step of the enzyme. The 3' DNA phosphoester ase activities of the wildtype and the suppressor mutant were also comparab le. No global change of the protein conformation is induced by the single o r double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type a nd suppressor mutant proteins have similar Mg2+ requirement for activity. T hese 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 pro tein. These results suggest a rather flexible conformation of the region su rrounding the metal binding site in hAPE-1 which is not obvious from the X- ray crystallographic structure. (C) 1999 Academic Press.