New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase
New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase
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TNP-AMP 与人肝果糖-1,6-双磷酸酶结合模式的新见解
DOI：
10.1016/j.saa.2016.04.002
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发表时间：
2016-08-05
影响因子：
4.4
通讯作者：
Wan, Jian
中科院分区：
文献类型：
--
作者：
Han, Xinya;Huang, Yunyuan;Wan, Jian
人肝果糖-1，6-二磷酸酶(FBPase)含有两个结合部位，即底物果糖-1，6-二磷酸(FBP)活性部位和一磷酸腺苷(AMP)变构部位。FBP活性部位通过稳定FBPase发挥作用，而变构部位通过与非底物分子结合而削弱FBPase的活性。荧光AMP类似物2‘，3’-O-(2，4，6-三硝基苯基)腺苷5‘-单磷酸(TNP-AMP)能竞争性地抑制AMP与变构中心的结合，因此可用于探索以变构中心为靶点的抑制剂的结合模式。在本研究中，我们重新研究了TNP-AMP与FBPase的结合方式。然而，我们目前的酶动力学分析表明，AMP和FBP都可以通过竞争FBPase来减少结合TNP-AMP的荧光，这表明TNP-AMP不仅与AMP变构部位结合，而且还与FBP活性部位结合。对位于FBP活性部位的K274L的诱变分析表明，K274残基对于TNP-AMP与FBPase活性部位的结合非常重要。结果进一步证明TNP-AMP能够单独与这两个位点结合。我们的研究为TNP-AMP与FBPase的结合机制提供了新的视角。TNP-AMP荧光探针可以用来检测分别被AMP和FBP饱和的FBPase或K247L突变体FBPase与抑制剂的结合部位(活性部位或变构部位)。(C)2016爱思唯尔B.V.保留所有权利。
Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase. (C) 2016 Elsevier B.V. All rights reserved.