title: Substrate binding by human apurinic/apyrimidinic endonuclease indicates a Briggs-Haldane mechanism.
abstract: Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5' 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 3'-terminus and a 28-mer with a phosphodeoxyribose at the 5'-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 3'-terminus resulted from beta-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 beta-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 k(on) and k(off) are 5 x 10(7) M(-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. k(off,product) >> 10 s(-1)). This scheme is in excellent agreement with the measured steady-state kinetic parameters.
authors.affiliation: Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555, USA.
authors.forename: P R
authors.initials: PR
authors.lastname: Strauss
authors.forename: W A
authors.initials: WA
authors.lastname: Beard
authors.forename: T A
authors.initials: TA
authors.lastname: Patterson
authors.forename: S H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008956:Models, Chemical; D011489:Protein Denaturation
chemical_list: D029968:Escherichia coli Proteins; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase