Analysis of human tyrosyl-DNA phosphodiesterase I catalytic residues.
Analysis of human tyrosyl-DNA phosphodiesterase I catalytic residues.
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人酪氨酰 DNA 磷酸二酯酶 I 催化残基的分析。
DOI：
10.1016/j.jmb.2004.03.013
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
BurginJr,AlexB
中科院分区：
文献类型：
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作者：
Raymond,AmyC;Rideout,MarcC;Staker,Bart;Hjerrild,Kathryn;BurginJr,AlexB
酪氨酸-DNA磷酸二酯酶I（Tdp 1）参与由拓扑异构酶I在体内产生的DNA损伤的修复。Tdp 1是磷脂酶D（PLD）超家族的一员，在体外水解3′-磷酸酪氨酰键产生3′-磷酸DNA和游离酪氨酸。在这里，我们使用合成的3′-（4-硝基）苯基，3′-（4-甲基）苯基，和3′-酪氨酸磷酸寡核苷酸来研究人类Tdp 1。人Tdp 1（hTdp 1）的动力学分析表明，该酶对所有三种底物具有纳摩尔亲和力，并且整体体外反应是扩散限制的。活性位点突变体使用这些修改后的基板的分析表明，hTdp 1使用酸/碱催化机制。结果表明，组氨酸493作为一般的酸在初始酯交换反应，在以前的晶体结构模型的基础上的假设一致。结果还表明，赖氨酸495和天冬酰胺516参与了一般的酸反应，晶体结构分析表明，这些残基可能在质子中继中起作用。与以前的晶体结构数据一起，新的功能数据提供了对所有PLD家族成员中发现的保守组氨酸、赖氨酸和天冬酰胺残基的机械理解。
Tyrosyl-DNA phosphodiesterase I (Tdp1) is involved in the repair of DNA lesions created by topoisomerase I in vivo. Tdp1 is a member of the phospholipase D (PLD) superfamily of enzymes and hydrolyzes 3′-phosphotyrosyl bonds to generate 3′-phosphate DNA and free tyrosine in vitro. Here, we use synthetic 3′-(4-nitro)phenyl, 3′-(4-methyl)phenyl, and 3′-tyrosine phosphate oligonucleotides to study human Tdp1. Kinetic analysis of human Tdp1 (hTdp1) shows that the enzyme has nanomolar affinity for all three substrates and the overall in vitro reaction is diffusion-limited. Analysis of active-site mutants using these modified substrates demonstrates that hTdp1 uses an acid/base catalytic mechanism. The results show that histidine 493 serves as the general acid during the initial transesterification, in agreement with hypotheses based on previous crystal structure models. The results also argue that lysine 495 and asparagine 516 participate in the general acid reaction, and the analysis of crystal structures suggests that these residues may function in a proton relay. Together with previous crystal structure data, the new functional data provide a mechanistic understanding of the conserved histidine, lysine and asparagine residues found among all PLD family members.