Pre-steady-state Kinetic Characterization of the AP Endonuclease Activity of Human AP Endonuclease 1

• Published in: The Journal of biological chemistry Vol. 282; no. 42; pp. 30577 - 30585
• Main Authors: Maher, Robyn L., Bloom, Linda B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.10.2007; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Binding Sites - genetics; Catalysis; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase - chemistry; DNA-(Apurinic or Apyrimidinic Site) Lyase - genetics; DNA-(Apurinic or Apyrimidinic Site) Lyase - metabolism; Humans; Hydrolysis; Kinetics; Mutation, Missense; Protein Binding - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M704341200

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.