Lindsley J E, Wang J C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
J Biol Chem. 1993 Apr 15;268(11):8096-104.
The initial rates of ATP hydrolysis and relaxation of negatively supercoiled DNA by highly purified wild-type and mutant yeast DNA topoisomerase II were measured under identical conditions to study the coupling between the ATPase activity of a type II DNA topoisomerase and its catalysis of the transport of one DNA segment through another. The results indicate that the binding of the enzyme to DNA stimulates its intrinsic ATPase activity by about 20-fold, and ATP binding to the pair of ATPase sites in a DNA-bound dimeric enzyme appears to be cooperative. The cooperativity in ATP binding may be significant in the coordination of the two halves of a DNA-bound enzyme dimer. At low ATP concentrations, the rate-limiting step in ATP usage appears to be slower than that in DNA transport, and DNA transport is relatively efficient in terms of ATP consumption: 1.9 +/- 0.5 ATP molecules are hydrolyzed/DNA transport event. At a saturating ATP concentration, however, there appears to be a reversal of these rate-limiting steps, and DNA transport is less efficient: 7.4 +/- 1.0 ATP molecules are hydrolyzed/DNA transport event. These data are interpreted in terms of a model in which a DNA-bound enzyme acts as an ATP-operated clamp for the capture and transport of a second DNA segment.
在相同条件下，测定了高度纯化的野生型和突变型酵母DNA拓扑异构酶II对负超螺旋DNA的ATP水解初始速率和松弛速率，以研究II型DNA拓扑异构酶的ATP酶活性与其催化一个DNA片段穿过另一个DNA片段的转运之间的偶联关系。结果表明，酶与DNA的结合使其内在ATP酶活性提高约20倍，并且ATP与结合在DNA上的二聚体酶中的一对ATP酶位点的结合似乎具有协同性。ATP结合的协同性可能在结合于DNA的酶二聚体两半部分的协调中具有重要意义。在低ATP浓度下，ATP使用的限速步骤似乎比DNA转运中的限速步骤慢，并且就ATP消耗而言，DNA转运相对高效：每发生一次DNA转运事件，有1.9±0.5个ATP分子被水解。然而，在ATP浓度饱和时，这些限速步骤似乎发生了逆转，并且DNA转运效率较低：每发生一次DNA转运事件，有7.4±1.0个ATP分子被水解。这些数据依据一个模型进行解释，在该模型中，结合于DNA的酶充当一个由ATP驱动的夹子，用于捕获和转运第二个DNA片段。
