A novel fluorescence assay to study propeptide interaction with gamma-glutamyl carboxylase.
A novel fluorescence assay to study propeptide interaction with gamma-glutamyl carboxylase.
复制标题
一种研究前肽与 γ-谷氨酰羧化酶相互作用的新型荧光测定法。
DOI：
10.1021/bi010332w
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Stafford,DW
中科院分区：
文献类型：
--
作者：
Presnell,SR;Tripathy,A;Lentz,BR;Jin,DY;Stafford,DW
维生素K依赖性γ-谷氨酰羧化酶催化其维生素K依赖性底物的选择谷氨酸残基翻译后修饰为γ-羧基谷氨酸。在这份报告中，我们描述了一种新的荧光分析，是敏感和特异性的活性羧化酶的前肽结合位点。我们采用该测定法进行了三个重要的观察：（1）紧密结合的荧光素标记的共有前肽可用于定量酶的活性部分。(2)荧光素标记的因子IX前肽的解离速率比羧化速率慢3000倍，这一差异可以解释羧化酶如何在同一结合事件期间对底物进行多次羧化。(3)我们发现的证据表明，底物结合到活性位点修改羧化酶的前肽结合位点。在存在和不存在其共底物的情况下前肽的解离速率的显著（9倍）差异可能代表大分子底物从酶的释放机制。此外，沉降速度和平衡实验表明酶与前肽的单体缔合。此外，羧化酶制剂在用于我们研究的缓冲液中是单分散的。
The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational modification of select glutamate residues of its vitamin K-dependent substrates to gamma-carboxyglutamate. In this report, we describe a new fluorescence assay that is sensitive and specific for the propeptide binding site of active carboxylase. We employed the assay to make three important observations: (1) A tight binding fluorescein-labeled consensus propeptide can be used to quantify the active fraction of the enzyme. (2) The off-rate for a fluorescein-labeled factor IX propeptide was 3000-fold slower than the rate of carboxylation, a difference that may explain how carboxylase can carry out multiple carboxylations of a substrate during the same binding event. (3) We show evidence that substrate binding to the active site modifies the propeptide binding site of carboxylase. The significant (9-fold) differences in off-rates for the propeptide in the presence and absence of its co-substrates may represent a release mechanism for macromolecular substrates from the enzyme. Additionally, sedimentation velocity and equilibrium experiments indicate a monomeric association of enzyme with propeptide. Furthermore, the carboxylase preparation is monodisperse in the buffer used for our studies.