Interaction between neuronal nitric-oxide synthase and tetrahydrobiopterin revisited: studies on the nature and mechanism of tight pterin binding.
Interaction between neuronal nitric-oxide synthase and tetrahydrobiopterin revisited: studies on the nature and mechanism of tight pterin binding.
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DOI：
10.1021/bi401307r
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发表时间：
2014-03-04
期刊：
影响因子：
2.9
通讯作者：
Gorren, Antonius C. F.
中科院分区：
文献类型：
--
作者：
Heine, Christian L.;Kolesnik, Bernd;Schmidt, Renate;Werner, Ernst R.;Mayer, Bernd;Gorren, Antonius C. F.
在杆状病毒感染的Sf9细胞中表达的重组神经元型一氧化氮合酶(NNOS)每个二聚体含有大约1个紧密结合的四氢生物蝶呤(BH4)，并结合第二个当量的BH4，解离常数在10-7-10-6M范围内。对于源自不产生BH4的大肠杆菌等表达系统的nNOS的蝶呤结合特性，人们知之甚少。我们通过监测它们对酶活性的影响，确定了大肠杆菌表达的nNOS与BH4和几种抑制性蝶呤的结合特性。原核表达的一氧化氮合酶被BH4单相激活，EC_(50)≈为2×10~(-7)M，缺乏紧密的蝶呤结合。然而，与BH4孵育一夜导致每个二聚体紧密结合一个BH4，产生一种类似于Sf9表达的nNOS的酶。与4-氨基四氢生物蝶呤预先孵育也能诱导紧密结合，但不能被7，8-二氢生物蝶呤或4-氨基二氢生物蝶呤诱导，这表明紧密结合部位的形成需要用完全还原的蝶啶预先孵育。动力学实验表明，在4℃下，1μM BH4需要大约10分钟才能形成紧密结合部位(1μM4-氨基BH4需要2分钟)，厌氧预培养实验表明O2不参与这一过程。凝胶电泳研究表明，紧结合位点的形成伴随着一氧化氮合酶二聚体强度的增加。我们认为，在一个涉及氧化还原化学的反应中，无蝶呤的nNOS与BH4孵育时，每个二聚体都会产生一个紧密的蝶呤结合位点，而另一个位点不受影响。
Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10–7–10–6 M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10–7 M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-dihydrobiopterin or 4-amino-dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 μM BH4 (2 min with 1 μM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry.
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影响因子：
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影响因子：
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DOI：
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发表时间：
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影响因子：
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作者：
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