Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen.
Evidence for the slow reaction of hypoxia-inducible factor prolyl hydroxylase 2 with oxygen.
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DOI：
10.1111/j.1742-4658.2010.07804.x
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发表时间：
2010-10
期刊：
影响因子：
--
通讯作者：
Schofield CJ
中科院分区：
文献类型：
--
作者：
Flashman E;Hoffart LM;Hamed RB;Bollinger JM Jr;Krebs C;Schofield CJ
动物对低氧的反应是由低氧诱导转录因子(HIF)介导的。人缺氧诱导因子受四种铁(II)和2-氧戊二酸(2OG)依赖的加氧酶的调节：脯氨酰羟基酶结构域酶(PhDS或EGLNS)1-3催化HIF中两个脯氨基残基的羟基化，触发其被蛋白酶体降解。缺氧诱导因子(FIH)催化缺氧诱导因子中天冬酰胺残基的羟化，抑制其转录活性。总体而言，HIF羟解酶负向调节HIF，以响应氧气浓度的增加。脯氨酸羟基酶结构域2(PHD2)是人类细胞中最重要的氧感受器，但其氧敏功能的动力学基础尚不清楚。我们报道了PHD2与氧气反应的分析。化学猝灭/质谱学实验表明，PHD2、Fe(II)、2OG和HIF-α的C端氧依赖降解结构域(CoDD)与氧反应生成羟化CodD和琥珀酸酯的反应速度比其他类似研究的2OG加氧酶慢得多(~100倍)。停流/紫外可见光谱实验表明，该反应产生了一个相对稳定的物种，在320 nm处有吸收；穆斯堡尔光谱实验表明，该物种可能不是像其他2OG加氧酶那样的Fe(IV)=O中间体。总体而言，结果表明，至少与其他研究的20G加氧酶相比，PHD2与氧的反应相对较慢，这一特性可能与其作为氧传感器的功能有关。
The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor (HIF). Human HIF is regulated by four Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases: Prolyl hydroxylase domain enzymes (PHDs or EGLNs) 1–3 catalyse hydroxylation of two prolyl-residues in HIF, triggering its degradation by the proteasome. Factor inhibiting HIF (FIH) catalyses hydroxylation of an asparagine-residue in HIF, inhibiting its transcriptional activity. Collectively, the HIF hydroxylases negatively regulate HIF in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 (PHD2) is the most important oxygen sensor in human cells; however the underlying kinetic basis of the oxygen sensing function of PHD2 is unclear. We report analyses of the reaction of PHD2 with oxygen. Chemical quench/mass spectrometry experiments showed that reaction of a complex of PHD2, Fe(II), 2OG and the C-terminal oxygen-dependent degradation domain of HIF-α (CODD) with oxygen to form hydroxylated CODD and succinate is much slower (~100 fold) than for other similarly studied 2OG oxygenases. Stopped flow/UV-visible spectroscopy experiments showed that the reaction produces a relatively stable species absorbing at 320nm; Mössbauer spectroscopic experiments implied that this species is likely not a Fe(IV)=O intermediate, as observed for other 2OG oxygenases. Overall the results suggest that, at least compared to other studied 2OG oxygenases, PHD2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor.
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