Measuring rates of ubiquitin chain formation as a functional readout of ligase activity.
Measuring rates of ubiquitin chain formation as a functional readout of ligase activity.
复制标题
测量泛素链形成的速率作为连接酶活性的功能读数。
DOI：
10.1007/978-1-61779-474-2_14
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Haas,ArthurL
中科院分区：
文献类型：
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作者：
Ronchi,VirginiaP;Haas,ArthurL
泛素结合途径内的特异性是由各组分之间的蛋白质结合亲和力来定义的。酶动力学提供了一种简单的高分辨率实验方法来定量这种蛋白质结合亲和性，并对酶催化反应的过渡状态产生了额外的机制见解。大多数泛素连接酶在缺乏同源靶蛋白的情况下以缓慢的速度形成自由的多泛素链，这是其整个催化循环的正常步骤。因此，多泛素链形成的速率可以作为一种动态报告功能来表征连接途径内的结合相互作用。我们描述了实验方法：(1)通过它们的化学计量形成125i -泛素硫酯来精确定量功能性E1和E2的浓度；(2)半定量筛选，根据泛素连接酶支持多泛素链形成的能力，确定其同源E2(s)；(3)定量研究kmandcata的初始速率，以衡量特定e2 -泛素硫酯底物支持连接酶催化的多泛素链形成的能力；(4)一种基于异肽酶的技术，用于区分在功能测定中形成的游离和共轭多泛素链。这些动力学方法提供了其他实验方法无法获得的机制见解，并且在表征蛋白质相互作用方面产生了超过其他技术的精度。
Specificity within the pathways of ubiquitin conjugation are defined by protein-binding affinities among the components. Enzyme kinetics provides a facile high-resolution experimental approach for quantitating such protein-binding affinities and yields additional mechanistic insights into the transition state of the enzyme-catalyzed reaction. Most ubiquitin ligases form free polyubiquitin chains at a slow rate in the absence of their cognate target protein as a normal step in their overall catalytic cycle. Rates of polyubiquitin chain formation can, therefore, be used as a reporter function kinetically to characterize binding interactions within the ligation pathway. We describe experimental approaches for: (1) precisely quantitating functional E1 and E2 concentrations by their stoichiometric formation of125I-ubiquitin thiolester; (2) semiquantitative screens to define the cognate E2(s) for ubiquitin ligases based on their ability to support polyubiquitin chain formation; (3) initial rate studies to quantifyKmandkcatas a measure of the ability of specific E2-ubiquitin thiolester substrates to support ligase-catalyzed polyubiquitin chain formation; and (4) an isopeptidase T-based technique for distinguishing between free and conjugated polyubiquitin chains formed in the functional assays. These kinetic methods provide mechanistic insights that are otherwise inaccessible by other experimental approaches and yield a precision in characterizing protein interactions that exceeds that of other techniques.