Transient-State Kinetics of Apurinic/Apyrimidinic (AP) Endonuclease 1 Acting on an Authentic AP Site and Commonly Used Substrate Analogs: The Effect of Diverse Metal Ions and Base Mismatches

Apurinic/apyrimidinic endonuclease 1 (APE1) is an Mg²⁺-dependent enzyme responsible for incising the DNA backbone 5′ to an apurinic/apyrimidinic (AP) site. Here, we use rapid quench flow (RQF) techniques to provide a comprehensive kinetic analysis of the strand-incision activity (k_chemistry) of APE1 acting on an authentic AP site along with two widely used analogs, a reduced AP site and a tetrahydrofuran (THF) site. In the presence of biologically relevant Mg²⁺, APE1 incises all three substrates at a rate faster than the resolution of the RQF, ≥700 s^–1. To obtain quantitative values of k_chemistry and to facilitate a comparison of the authentic substrate versus the substrate analogs, we replaced Mg²⁺ with Mn²⁺ or Ni²⁺ or introduced a mismatch 5′ to the lesion site. Both strategies were sufficient to slow k_chemistry and resulted in rates within the resolution of the RQF. In all cases where quantitative rates were obtained, k_chemistry for the reduced AP site is indistinguishable from the authentic AP site. Notably, there is a small decrease, ∼1.5-fold, in k_chemistry for the THF site relative to the authentic AP site. These results highlight a role in strand incision for the C1′ oxygen of the AP site and warrant consideration when designing experiments using substrate analogs.