Department of Chemistry, University of Massachusetts, Amherst, United States.
Department of Chemistry, University of Massachusetts, Amherst, United States.
J Inorg Biochem. 2018 Jan;178:63-69. doi: 10.1016/j.jinorgbio.2017.10.001. Epub 2017 Oct 7.
Non-heme Fe(II)/α-ketoglutarate (αKG)-dependent oxygenases catalyze a wide array of reactions through coupling oxidative decarboxylation of αKG to substrate oxygenation. This class of enzymes follows a sequential mechanism in which O reacts only after binding primary substrate, raising questions over how protein structure tailors molecular access to the Fe(II) cofactor. The enzyme "factor inhibiting hypoxia inducible factor" (FIH) senses pO in human cells by hydroxylating the C-terminal transactivation domain (CTAD), suggesting that structural elements limiting molecular access to the active site may limit the pO response. In this study, we tested the impact of a solvent-accessible tunnel in FIH on molecular access to the active site in FIH. The size of the tunnel was increased through alanine point mutagenesis (Y93A, E105A, and Q147A), followed by a suite of mechanistic and spectroscopic probes. Steady-state kinetics varying O or CTAD indicated that O passage through the tunnel was not affected by Ala substitutions, allowing us to conclude that this narrow tunnel did not impact pO sensing by FIH. Steady-state kinetics with varied αKG concentrations revealed increased substrate inhibition for the Ala variants, suggesting that a second αKG molecule may bind near the active site of FIH. If this solvent-accessible tunnel is the O entry tunnel, it may be narrow in order to permit O access while preventing metabolic intermediates, such as αKG, from inhibiting FIH under physiological conditions.
非血红素 Fe(II)/α-酮戊二酸 (αKG)-依赖性加氧酶通过将 αKG 的氧化脱羧与底物的氧化偶联来催化广泛的反应。这类酶遵循顺序机制，其中 O 仅在结合主要底物后才反应，这引发了关于蛋白质结构如何调整分子对 Fe(II)辅因子的接近的问题。酶“缺氧诱导因子抑制因子”(FIH)通过羟基化 C 端转录激活结构域 (CTAD)来感知人细胞中的 pO，这表明限制分子接近活性位点的结构元件可能会限制 pO 反应。在这项研究中，我们测试了 FIH 中溶剂可及隧道对 FIH 活性位点分子接近的影响。通过丙氨酸点突变（Y93A、E105A 和 Q147A）增加隧道的大小，然后进行一系列的机制和光谱探针研究。O 或 CTAD 的稳态动力学表明，隧道中的 Ala 取代物不影响 O 通过，这使我们能够得出结论，这个狭窄的隧道不会影响 FIH 的 pO 感应。用不同的 αKG 浓度进行的稳态动力学揭示了 Ala 变体的底物抑制增加，这表明第二个 αKG 分子可能在 FIH 的活性位点附近结合。如果这个溶剂可及的隧道是 O 进入隧道，那么它可能很窄，以便在防止代谢中间产物（如 αKG）在生理条件下抑制 FIH 的同时，允许 O 进入。
