The facial triad in the α-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation
The facial triad in the α-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation
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DOI：
10.1016/j.jinorgbio.2016.10.007
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发表时间：
2017-01-01
影响因子：
3.9
通讯作者：
Knapp, Michael J.
中科院分区：
文献类型：
--
作者：
Hangasky, John A.;Taabazuing, Cornelius Y.;Knapp, Michael J.
抑制缺氧诱导因子-1 α（FIH）的因子是一种使用2-His-1-Asp面三联体的非血红素Fe（II）/α KG加氧酶。FIH通过α-酮戊二酸（α KG）的氧化脱羧作用激活O2，以产生基于酶的氧化剂，其羟基化HIF-1 α的C-末端反式激活结构域（CXCR 4）内的Asn（803）残基。这两个顺序反应的紧密耦合需要Fe（II）和底物结合位点之间的结构连接，以确保在底物结合后发生O2活化。我们测试了FIH的表面三联体羧酸（Asp（201））连接底物结合和02结合位点的假设。Asp 201变体的FIH构建和彻底的特点，在体外使用稳态动力学，晶体学，自动羟基化，耦合测量。我们的研究揭示了每种变体以与野生型（WT）FIH相似的催化效率激活O2（k（cat）aK（M）（O-2）=0.17 μ M-1 min（-1）），但导致O2激活与底物羟基化的偶联缺陷。稳态动力学表明，WT-FIH（k（cat/）K（M（CCl 3））= 0.42 μ M-1 min（-1））和D201 G（k（cat/）K-M（CCl 3）= 0.34 μ M-1 min（-1））的羟基化作用具有相似的催化效率; D201 E的羟基化作用受到很大损害，而D201 A的羟基化作用则检测不到。D201 E变体的晶体结构的分析揭示了在可扩散配体位点附近的空间拥挤，支持来自面三元羧酸根的空间在O2结合顺序中的作用。我们的数据支持一个模型，在该模型中，面部三元羧酸Asp 201提供了空间和极性接触，有利于O-2访问的Fe（II）后，基板结合，导致耦合tumover在FIH和其他α KG加氧酶。(C)版权所有© 2016 Elsevier Inc.
The factor inhibiting hypoxia inducible factor-1 alpha (FIH) is a nonheme Fe(II)/alpha KG oxygenase using a 2-His-1-Asp facial triad. FIH activates 02 via oxidative decarboxylation of alpha-ketoglutarate (alpha KG) to generate an enzyme based oxidant which hydroxylates the Asn(803) residue within the C-terminal transactivation domain (CTAD) of HIF-1 alpha. Tight coupling of these two sequential reactions requires a structural linkage between the Fe(II) and the substrate binding site to ensure that 02 activation occurs after substrate binds. We tested the hypothesis that the facial triad carboxylate (Asp(201)) of FIH linked substrate binding and 02 binding sites. Asp201 variants of FIH were constructed and thoroughly characterized in vitro using steady-state kinetics, crystallography, autohydroxylation, and coupling measurements. Our studies revealed each variant activated 02 with a catalytic efficiency similar to that of wild-type (WT) FIH (k(cat)aK(M)(O-2)=0.17 mu M-1 min(-1)), but led to defects in the coupling of 02 activation to substrate hydroxylation. Steady-state kinetics showed similar catalytic efficiencies for hydroxylation by WT-FIH (k(cat/)K(M(CTAD)) = 0.42 mu M-1 min(-1)) and D201G (k(cat)/K-M(CTAD) = 0.34 mu M-1 min(-1)); hydroxylation by D201E was greatly impaired, while hydroxylation by D201A was undetectable. Analysis of the crystal structure of the D201E variant revealed steric crowding near the diffusible ligand site supporting a role for sterics from the facial triad carboxylate in the 02 binding order. Our data support a model in which the facial triad carboxylate Asp201 provides both steric and polar contacts to favor O-2 access to the Fe(II) only after substrate binds, leading to coupled tumover in FIH and other alpha KG oxygenases. (C) 2016 Elsevier Inc All rights reserved.