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. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.