Mechanism of HIV Reverse Transcriptase Inhibition by Zinc FORMATION OF A HIGHLY STABLE ENZYME-(PRIMER-TEMPLATE) COMPLEX WITH PROFOUNDLY DIMINISHED CATALYTIC ACTIVITY
Mechanism of HIV Reverse Transcriptase Inhibition by Zinc FORMATION OF A HIGHLY STABLE ENZYME-(PRIMER-TEMPLATE) COMPLEX WITH PROFOUNDLY DIMINISHED CATALYTIC ACTIVITY
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DOI：
10.1074/jbc.m111.289850
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发表时间：
2011-11-25
影响因子：
4.8
通讯作者：
DeStefano, Jeffrey J.
中科院分区：
文献类型：
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作者：
Fenstermacher, Katherine J.;DeStefano, Jeffrey J.
几个生理相关的阳离子，包括Ca 2+，Mn 2+，和Zn 2+已被证明抑制HIV逆转录酶（RT），大概是通过竞争性取代一个或多个镁离子结合到RT。我们分析了Zn 2+对逆转录的影响，并将其与Ca 2+和Mn 2+进行了比较。使用核苷酸延伸效率作为读数，Zn 2+显示出与2 mM Mg 2+的反应的显著抑制，即使当仅以类似于5 μ M存在时。Mn ~（2+）和Ca ~（2+）也有抑制作用，但浓度较高。Mn 2+和Zn 2+（但不是Ca 2+）两者在不存在Mg 2+的情况下支持RT掺入，其中Mn 2+更有效。Zn 2+、Mg 2+和Mg 2+的最大延伸速率分别为每秒0.1、1和3.5个核苷酸。锌在类似于25 μ M时支持最佳RNase H活性，类似于在低dNTP浓度存在下核苷酸添加的最佳活性。令人惊讶的是，在逆转录过程中的持续合成能力（与酶结合的单个结合事件中掺入的核苷酸的平均数量）与Zn 2+和Mg 2+相当，并且单个RT分子能够在Zn 2+存在下在相同模板上持续延伸数小时。与该结果一致，RT-Zn 2 +-（引物-模板）复合物的半衰期为220 +/-60分钟，而Mg 2+的半衰期仅为1.7 +/-1分钟，表明与Zn 2+的结合类似于130倍更稳定。基本上，Zn 2+的存在促进高度稳定的缓慢进展RT-（引物-模板）复合物的形成。
Several physiologically relevant cations including Ca2+, Mn2+, and Zn2+ have been shown to inhibit HIV reverse transcriptase (RT), presumably by competitively displacing one or more Mg2+ ions bound to RT. We analyzed the effects of Zn2+ on reverse transcription and compared them to Ca2+ and Mn2+. Using nucleotide extension efficiency as a readout, Zn2+ showed significant inhibition of reactions with 2 mM Mg2+, even when present at only similar to 5 mu M. Mn2+ and Ca2+ were also inhibitory but at higher concentrations. Both Mn2+ and Zn2+ (but not Ca2+) supported RT incorporation in the absence of Mg2+ with Mn2+ being much more efficient. The maximum extension rates with Zn2+, Mg2+, and Mg2+ were similar to 0.1, 1, and 3.5 nucleotides per second, respectively. Zinc supported optimal RNase H activity at similar to 25 mu M, similar to the optimal for nucleotide addition in the presence of low dNTP concentrations. Surprisingly, processivity (average number of nucleotides incorporated in a single binding event with enzyme) during reverse transcription was comparable with Zn2+ and Mg2+, and single RT molecules were able to continue extension in the presence of Zn2+ for several hours on the same template. Consistent with this result, the half-life for RT-Zn2+-(primer-template) complexes was 220 +/- 60 min and only 1.7 +/- 1 min with Mg2+, indicating similar to 130-fold more stable binding with Zn2+. Essentially, the presence of Zn2+ promotes the formation of a highly stable slowly progressing RT-(primer-template) complex.