Podust V N, Korobeĭnicheva T O, Nevinskiĭ G A, Rikhter V A, Abramova T I, Lavrik O I
Bioorg Khim. 1990 Feb;16(2):226-35.
Modification of the human placenta DNA polymerase alpha by 2',3'-epoxyadenosine 5'-triphosphate (eATP) was investigated. The latter binds to the protein both in absence and in presence of template-primer complex. However for inactivation of the enzyme, reagent-complementary template, primer and Me2(+)-ions are required. The inactivation is apparently due to the affinity modification of dNTP-binding site by eATP; covalent binding of the reagent off the enzyme's active site without affecting the DNA polymerase activity is also suggested. The enzyme inactivation by eATP and its protection from inactivation in the presence of dATP were used to determine Kd values of complexes of the enzyme with eATP (90 microM) and dATP (1 microM), the latter value being 13-times lower than Km for dATP (13 microM) in the polymerisation reaction. Using the dependence of the DNA polymerase inactivation by eATP on the primer concentration, Kd for enzyme-primer complexes were estimated. The Kd value for d(pA)10 (0.33 microM) was close to Km value (0.43 microM) for this primer. eATP was concluded to be a useful reagent for estimating the efficiency of the complex formation of different ligands with dNTP- and primer-binding sites of DNA polymerase.
研究了2',3'-环氧腺苷5'-三磷酸（eATP）对人胎盘DNA聚合酶α的修饰作用。无论有无模板引物复合物，后者均能与该蛋白结合。然而，要使该酶失活，需要试剂互补的模板、引物和Me2(+)离子。失活显然是由于eATP对dNTP结合位点的亲和修饰；也有人提出试剂在酶活性位点之外的共价结合不会影响DNA聚合酶的活性。利用eATP使酶失活以及在dATP存在下对其失活的保护作用来测定酶与eATP（90微摩尔）和dATP（1微摩尔）复合物的Kd值，后者的值比聚合反应中dATP（13微摩尔）的Km值低13倍。利用eATP使DNA聚合酶失活对引物浓度的依赖性，估算了酶 - 引物复合物的Kd值。d(pA)10（0.33微摩尔）的Kd值与该引物的Km值（0.43微摩尔）相近。得出结论，eATP是一种用于评估不同配体与DNA聚合酶的dNTP和引物结合位点形成复合物效率的有用试剂。
