Hammonds T R, Maxwell A
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
J Biol Chem. 1997 Dec 19;272(51):32696-703. doi: 10.1074/jbc.272.51.32696.
We have purified human topoisomerase IIalpha from HeLa cells and studied its ATPase reaction. The ATPase activity is stimulated by DNA and shows apparent Michaelis-Menten kinetics. Although the ATPase activity of human topoisomerase IIalpha is lower than that of Saccharomyces cerevisiae, it is more active in decatenation, implying more efficient coupling of the ATPase to DNA strand passage under these conditions. Using plasmid pBR322 as the DNA cofactor, the reaction shows hyperstimulation by DNA at a base pair to enzyme dimer ratio of 100-200:1. When DNA fragments are used as the cofactor, the reaction requires > approximately 100 base pairs to stimulate the activity and fragments of approximately 300 base pairs show hyperstimulation. This behavior can be rationalized in terms of the enzyme requiring fragments that can bind to both the DNA gate and the ATP-operated clamp in order for the ATPase reaction to be stimulated. Hyperstimulation is a consequence of the saturation of DNA with enzyme. The mechanistic implications of these results are discussed.
我们从HeLa细胞中纯化了人拓扑异构酶IIα，并研究了其ATP酶反应。DNA可刺激ATP酶活性，并呈现出明显的米氏动力学。尽管人拓扑异构酶IIα的ATP酶活性低于酿酒酵母，但它在解连环反应中更具活性，这意味着在这些条件下ATP酶与DNA链通过的偶联更有效。以质粒pBR322作为DNA辅因子，当碱基对与酶二聚体的比例为100 - 200:1时，反应显示出DNA的超刺激作用。当使用DNA片段作为辅因子时，反应需要大于约100个碱基对来刺激活性，约300个碱基对的片段显示出超刺激作用。这种行为可以从酶需要能够与DNA门和ATP操作钳都结合的片段才能刺激ATP酶反应的角度来解释。超刺激是酶使DNA饱和的结果。讨论了这些结果的机制意义。
