Prasad R, Beard W A, Strauss P R, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555, USA.
J Biol Chem. 1998 Jun 12;273(24):15263-70. doi: 10.1074/jbc.273.24.15263.
DNA polymerase beta (beta-pol) cleaves the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5'-incised by AP endonuclease, resulting in a 5' 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of beta-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of beta-pol are similar. However, kcat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5' dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of beta-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5'-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.
DNA聚合酶β（β-pol）可切割完整无嘌呤/无嘧啶（AP）位点3'端的磷酸二酯键（即AP裂解酶活性）。即使AP位点先前已被AP核酸内切酶在5'端切割，同一键仍会被切割，从而产生5' 2-脱氧核糖5-磷酸（即dRP裂解酶活性）。我们通过稳态动力学对β-pol的氨基末端8 kDa结构域和整个39 kDa聚合酶的这些裂解反应进行了表征。稳态动力学分析表明，β-pol的dRP裂解酶活性和AP裂解酶活性的米氏常数相似。然而，完整AP位点上AP裂解酶活性的催化常数（kcat）比AP核酸内切酶预切割位点上的低约200倍。8 kDa结构域在完整AP位点上的效率也低于预切割位点。在没有Mg2+的情况下，全长酶和8 kDa结构域能有效地从预切割的AP位点去除5' dRP，β-pol和8 kDa结构域dRP裂解酶催化效率的pH曲线显示出较宽的碱性pH最佳值。磷酸吡哆醛对dRP裂解酶活性的抑制作用与在裂解酶化学反应中作为席夫碱亲核试剂的伯胺（Lys72）的参与一致。
