SUBSTRATE-BINDING BY HUMAN APURINIC APYRIMIDINIC ENDONUCLEASE INDICATES A BRIGGS-HALDANE MECHANISM/

Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5' to a DNA abasic site, We have characterized this reaction by steady-stat e and transient-state kinetics with purified human AP endo, which had been expressed in Escherichia coli, The substrate was a 49-base pair o ligonucleotide 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 pr oducts of the AP endo nicking reaction were a 20-mer with a hydroxyl g roup 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 glycosylas e with a reducing agent, Otherwise, a 20-mer with phosphoribose at the 3'-terminus resulted from beta-elimination. In agreement with others, K-m and k(cat) were 100 nar and 10 s(-1), respectively. Heat treatmen t of the abasic site-containing 49-mer without enzyme also resulted in conversion to the beta-elimination product, The resultant heat degrad ation product was an efficient inhibitor of AP endo with a K-i of 30 n M. The enzyme required divalent cation (Mg2+) for activity, but bound substrate DNA in the absence of Mg2+. Electrophoretic mobility shift a ssays indicated that AP endo bound tightly to DNA containing an abasic site and formed a 1:1 complex at low enzyme concentrations, The assoc iation and dissociation rate constants for substrate binding to AP end o were determined by using a challenge assay to follow AP endo-substra te complex formation, Heat degradation product together with heparin s erved as an effective trap for free enzyme, The results are consistent with a Briggs-Haldane mechanism where k(om) and k(off) are 5 x 10(7) M(-1) s(-1) and 0.04 s(-1), respectively (K-d = 0.8 nM), k(cat) 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-s tate kinetic parameters.