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.
Department of Chemistry, Brown University , 324 Brook Street, Providence, Rhode Island 02912, United States.
Kelly M
KM
Schermerhorn
Sarah
S
Delaney
D020137:Base Pair Mismatch; D001665:Binding Sites; D002614:Chelating Agents / Q000494:pharmacology; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism*; D059372:Nucleotide Motifs; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D002614:Chelating Agents; D008024:Ligands; D011994:Recombinant Proteins; D008345:Manganese; D009532:Nickel; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium