Substrate Binding by Human Apurinic/Apyrimidinic Endonuclease Indicates a Briggs-Haldane Mechanism

Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5ʹ to a DNA abasic site. We have characterized this reaction by steady-state and transient-state kinetics with purified human AP endo, which had been expressed in Escherichia coli. The substrate was a 49-base pair oligonucleotide with an abasic site at position 21. This substrate was generated by treating a 49-mer duplex oligonucleotide with a single G/U located at position 21 with uracil-DNA glycosylase. The enzymatic products of the AP endo nicking reaction were a 20-mer with a hydroxyl group at the 3ʹ-terminus and a 28-mer with a phosphodeoxyribose at the 5ʹ-terminus. To obtain maximal enzymatic activity, it was necessary to stabilize the abasic site during treatment with uracil-DNA glycosylase with a reducing agent. Otherwise, a 20-mer with phosphoribose at the 3ʹ-terminus resulted from β-elimination. In agreement with others, Km and kcat were 100 nM and 10 s-1, respectively. Heat treatment of the abasic site-containing 49-mer without enzyme also resulted in conversion to the β-elimination product. The resultant heat degradation product was an efficient inhibitor of AP endo with a Ki of 30 nM. The enzyme required divalent cation (Mg2+) for activity, but bound substrate DNA in the absence of Mg2+. Electrophoretic mobility shift assays indicated that AP endo bound tightly to DNA containing an abasic site and formed a 1:1 complex at low enzyme concentrations. The association and dissociation rate constants for substrate binding to AP endo were determined by using a challenge assay to follow AP endo-substrate complex formation. Heat degradation product together with heparin served as an effective trap for free enzyme. The results are consistent with a Briggs-Haldane mechanism where kon and koff are 5 × 107M-1 s-1 and 0.04 s-1, respectively (Kd = 0.8 nM), kcat is 10 s-1, and product release is very rapid (i.e. koff,product≫ 10 s-1). This scheme is in excellent agreement with the measured steady-state kinetic parameters. Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5ʹ to a DNA abasic site. We have characterized this reaction by steady-state and transient-state kinetics with purified human AP endo, which had been expressed in Escherichia coli. The substrate was a 49-base pair oligonucleotide with an abasic site at position 21. This substrate was generated by treating a 49-mer duplex oligonucleotide with a single G/U located at position 21 with uracil-DNA glycosylase. The enzymatic products of the AP endo nicking reaction were a 20-mer with a hydroxyl group at the 3ʹ-terminus and a 28-mer with a phosphodeoxyribose at the 5ʹ-terminus. To obtain maximal enzymatic activity, it was necessary to stabilize the abasic site during treatment with uracil-DNA glycosylase with a reducing agent. Otherwise, a 20-mer with phosphoribose at the 3ʹ-terminus resulted from β-elimination. In agreement with others, Km and kcat were 100 nM and 10 s-1, respectively. Heat treatment of the abasic site-containing 49-mer without enzyme also resulted in conversion to the β-elimination product. The resultant heat degradation product was an efficient inhibitor of AP endo with a Ki of 30 nM. The enzyme required divalent cation (Mg2+) for activity, but bound substrate DNA in the absence of Mg2+. Electrophoretic mobility shift assays indicated that AP endo bound tightly to DNA containing an abasic site and formed a 1:1 complex at low enzyme concentrations. The association and dissociation rate constants for substrate binding to AP endo were determined by using a challenge assay to follow AP endo-substrate complex formation. Heat degradation product together with heparin served as an effective trap for free enzyme. The results are consistent with a Briggs-Haldane mechanism where kon and koff are 5 × 107M-1 s-1 and 0.04 s-1, respectively (Kd = 0.8 nM), kcat is 10 s-1, and product release is very rapid (i.e. koff,product≫ 10 s-1). This scheme is in excellent agreement with the measured steady-state kinetic parameters.