Measurement of internal substrate binding in dehaloperoxidase-hemoglobin by competition with the heme-fluoride binding equilibrium.
Measurement of internal substrate binding in dehaloperoxidase-hemoglobin by competition with the heme-fluoride binding equilibrium.
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通过与血红素-氟化物结合平衡的竞争来测量脱卤过氧化物酶-血红蛋白中的内部底物结合。
DOI：
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发表时间：
2015
影响因子：
3.3
通讯作者：
S. Franzen
中科院分区：
文献类型：
--
作者：
Jing Zhao;J. Moretto;Peter Le;S. Franzen
以氟离子为探针，研究了装饰双沟藻脱卤过氧化物酶-血红蛋白（DHP）的内部底物结合。通过应用氟化物滴定策略，使用紫外-可见光谱，我们已经研究了一系列的卤代苯酚，其他取代的苯酚，卤代吲哚，和几种天然氨基酸，结合内部（和非共价）的远端结合口袋的血红素。这种方法已经确定2，4-二溴苯酚（2，4-DBP）是迄今为止发现的最紧密的结合底物，其结合亲和力比4-溴苯酚（4-BP）（DHP中已知的内部结合抑制剂）的结合亲和力紧密约20倍。结合共振拉曼光谱，我们证实了氟阴离子与内结合分子之间存在竞争结合平衡。我们进一步研究了稳定外源性氟化物配体的DHP活性位点的氢键网络。这些测量结果证明了一个通用的方法，用于确定底物结合亲和力的差异的基础上检测的竞争性氟化物结合平衡。当考虑到结构和机理数据时，2，4-二溴苯酚比任何其他底物结合更紧密的结合的重要性是显而易见的。
The application of fluoride anion as a probe for investigating the internal substrate binding has been developed and applied to dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata. By applying the fluoride titration strategy using UV-vis spectroscopy, we have studied series of halogenated phenols, other substituted phenols, halogenated indoles, and several natural amino acids that bind internally (and noncovalently) in the distal binding pocket of the heme. This approach has identified 2,4-dibromophenol (2,4-DBP) as the tightest binding substrate discovered thus far, with approximately 20-fold tighter binding affinity than that of 4-bromophenol (4-BP), a known internally binding inhibitor in DHP. Combined with resonance Raman spectroscopy, we have confirmed that competitive binding equilibria exist between fluoride anion and internally bound molecules. We have further investigated the hydrogen bonding network of the active site of DHP that stabilizes the exogenous fluoride ligand. These measurements demonstrate a general method for determination of differences in substrate binding affinity based on detection of a competitive fluoride binding equilibrium. The significance of the binding that 2,4-dibromophenol binds more tightly than any other substrate is evident when the structural and mechanistic data are taken into consideration.
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DOI：
10.1016/j.bbrc.2012.12.047
发表时间：
2013-01
影响因子：
3.1
作者：
G. Schkolnik;T. Utesch;Junjie Zhao;Shu Jiang;M. Thompson;M. Mroginski;P. Hildebrandt;S. Franzen
通讯作者：
G. Schkolnik;T. Utesch;Junjie Zhao;Shu Jiang;M. Thompson;M. Mroginski;P. Hildebrandt;S. Franzen
影响因子：
20.6
作者：
Spiro TG;Soldatova AV;Balakrishnan G
通讯作者：
Balakrishnan G
影响因子：
2.9
作者：
BERRY, EA;TRUMPOWER, BL
通讯作者：
TRUMPOWER, BL
影响因子：
15
作者：
McWhirter, Claire;Lund, Elizabeth A.;Tanifum, Eric A.;Feng, Guoqiang;Sheikh, Qaiser I.;Hengge, Alvan C.;Williams, Nicholas H.
通讯作者：
Williams, Nicholas H.
脱卤过氧化物酶的复合 ES 通过两种替代途径衰减，具体取决于远端组氨酸的构象。
影响因子：
15
作者：
Thompson MK
通讯作者：
Thompson MK