Steady-state and rapid kinetic analysis of topoisomerase II trapped as the closed-clamp intermediate by ICRF-193
Steady-state and rapid kinetic analysis of topoisomerase II trapped as the closed-clamp intermediate by ICRF-193
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DOI：
10.1074/jbc.275.4.2613
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发表时间：
2000-01-28
影响因子：
4.8
通讯作者：
Lindsley, JE
中科院分区：
文献类型：
--
作者：
Morris, SK;Baird, CL;Lindsley, JE
DNA拓扑异构酶II使用ATP水解的复杂的顺序机制来催化一个DNA双链体通过另一个DNA双链体中的瞬时断裂的运输。ICRF-193是拓扑异构酶II的催化抑制剂，已知其捕获该酶的闭合钳中间体形式，使用稳态和快速动力学ATP酶和DNA转运测定，我们分析了药物如何捕获该中间体扰乱拓扑异构酶II机制。该药物对第一次脱连环转化率没有影响，但有效抑制随后的转化率，对酿酒酵母酶的IC 50为6.5 +/- 1 μ w。该药物通过一种不寻常的混合型机制抑制拓扑异构酶II的ATP酶活性;该药物不是ATP的竞争性抑制剂，即使在饱和浓度的药物下，该酶仍继续水解ATP，尽管速率降低。以药物结合的闭合钳形式特异性分离的拓扑异构酶II继续水解ATP，表明酶钳不需要重新打开以结合和水解ATP。当通过加入ATP启动快速淬灭ATP酶测定时，药物对第一或第二ATP的顺序水解没有影响。相反，当药物预先结合时，该酶以不受抑制的速率水解一个标记的ATP，但不水解第二个ATP。这些结果根据拓扑异构酶II的催化机制解释，并表明ICRF-193与结合到一个ADP的酶相互作用。
DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme, Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC50 of 6.5 +/- 1 mu w for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP, When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP, By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP, These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.