title: Pre-steady-state kinetic characterization of the AP endonuclease activity of human AP endonuclease 1.
abstract: Human AP endonuclease 1 (APE1, REF1) functions within the base excision repair pathway by catalyzing the hydrolysis of the phosphodiester bond 5 ' to a baseless sugar (apurinic or apyrimidinic site). The AP endonuclease activity of this enzyme and two active site mutants were characterized using equilibrium binding and pre-steady-state kinetic techniques. Wild-type APE1 is a remarkably potent endonuclease and highly efficient enzyme. Incision 5 ' to AP sites is so fast that a maximal single-turnover rate could not be measured using rapid mixing/quench techniques and is at least 850 s(-1). The entire catalytic cycle is limited by a slow step that follows chemistry and generates a steady-state incision rate of about 2 s(-1). Site-directed mutation of His-309 to Asn and Asp-210 to Ala reduced the single turnover rate of incision 5 ' to AP sites by at least 5 orders of magnitude such that chemistry (or a step following DNA binding and preceding chemistry) and not a step following chemistry became rate-limiting. Our results suggest that the efficiency with which APE1 can process an AP site in vivo is limited by the rate at which it diffuses to the site and that a slow step after chemistry may prevent APE1 from leaving the site of damage before the next enzyme arrives to continue the repair process.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245, USA.
authors.forename: Robyn L
authors.initials: RL
authors.lastname: Maher
authors.forename: Linda B
authors.initials: LB
authors.lastname: Bloom
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics
chemical_list: D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase