Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate
Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate
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DOI：
10.1074/jbc.275.20.15246
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发表时间：
2000-05-19
影响因子：
4.8
通讯作者：
Bjornsti, MA
中科院分区：
文献类型：
--
作者：
Fertala, J;Vance, JR;Bjornsti, MA
真核生物DNA拓扑异构酶I （Top1p）催化DNA拓扑结构的变化，是喜树碱的细胞靶点，最近的酶结构报道强调了活性位点酪氨酸的保守氨基酸n端的重要性，以及Asn-726在介导Top1p对喜树碱敏感性中的作用。为了研究该残基对酶催化的贡献，我们评估了用His、Asp或Ser取代Asn-726对酵母Top1p的影响。Top1N726S和Top1N726D突变蛋白对喜树碱具有抗性，尽管Ser突变蛋白的特征是缺乏可检测到的活性变化。因此，喜树碱的细胞毒性需要一个紧邻活性位点酪氨酸的n端基本残基。然而，用Asp或His代替Asn-726会干扰催化循环的不同方面，导致细胞致死。与喜树碱抑制酶催化的DNA结合相比，His取代基提高了DNA的断裂速度，而Asp突变则降低了DNA的酶结合速度。然而，这些对酶催化的影响并不相互排斥，因为他的突变体对喜树碱过敏。这些结果表明，在开发能够靶向Top1p催化循环不同方面的抗肿瘤药物时，可能会探索中毒DNA拓扑异构酶I的不同机制。
Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin, Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin, To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p, Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity. Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity. However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA. Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin. These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle.