(18)O Kinetic isotope effects in G protein GTPases

(18)O G 蛋白 GTP 酶中的动力学同位素效应

基本信息

批准号：
7086181
负责人：
Stephen R Sprang
金额：
$ 22.09万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2008-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The goal of the proposed research is to define the transition state and mechanism for G protein-catalyzed GTP hydrolysis. G proteins, which include members of the Ras superfamily and the alpha subunits of heterotrimeric G proteins (Galpha), are signal transducers when bound to GTP, but are inactivated by their intrinsic or GTPase activity. In vivo, GTPase activity can be accelerated by GTPase Activating Proteins. Intrinsic GTPase activity is slow and conformational changes in the catalytic site may be rate-limiting. GAP proteins accelerate intrinsic GTPase rates both by reducing the kinetic barrier to conformational changes in the G protein catalytic site and by facilitating chemical steps in the reaction. GAPs for the small G proteins Ras and Ran, and for the heterotrimeric Galpha proteins, appear to accelerate the GTPase activity of their G protein substrates by different mechanisms. Of particular interest is whether GAP proteins change the transition state for G protein-catalyzed GTPase reactions. It is proposed to measure the Kinetic Isotope Effect (KIE) for hydrolysis of specifically (18)O-labeled GTP substrates in order to determine whether conformational or chemical steps are rate-limiting for G protein and GAP-facilitated GTP hydrolysis. Primary and secondary isotope effects will be measured to determine the nature of the transition states for GTP hydrolysis catalyzed by Ras, Ran and Galpha-i1, in the presence of appropriate nucleotide exchange factors and GAPs. KIEs for GTPase reactions catalyzed by Galpha-i1 containing active site mutations shall be conducted in order to probe the role of catalytic residues in transition-state formation. A sensitive Chemical Reaction Interface coupled to an Isotope Ratio Mass Spectrometer shall be used to determine precisely the relative isotopic substitution of remotely (13)C-labeled (18)O-GTP substrates and products. The proposed methodology requires only nanomolar quantities of isotopically labeled substrates and is therefore applicable to mechanistic sudies of variety of ATPases and GTPases of significant biochemical and physiological interest.

描述（由申请人提供）：本研究的目标是确定 G 蛋白催化 GTP 水解的过渡态和机制。 G 蛋白包括 Ras 超家族的成员和异源三聚体 G 蛋白 (Gα) 的 α 亚基，当与 GTP 结合时是信号转导器，但会因其内在活性或 GTP 酶活性而失活。在体内，GTP酶活性可以通过GTP酶激活蛋白来加速。内在的 GTP 酶活性很慢，催化位点的构象变化可能会限制速率。 GAP 蛋白通过减少 G 蛋白催化位点构象变化的动力学障碍和促进反应中的化学步骤来加速内在 GTP 酶速率。小 G 蛋白 Ras 和 Ran 以及异源三聚体 Galpha 蛋白的 GAP 似乎通过不同的机制加速其 G 蛋白底物的 GTP 酶活性。特别令人感兴趣的是 GAP 蛋白是否会改变 G 蛋白催化的 GTP 酶反应的过渡态。建议测量特定 (18)O 标记的 GTP 底物水解的动力学同位素效应 (KIE)，以确定构象步骤或化学步骤是否限制 G 蛋白和 GAP 促进的 GTP 水解。在适当的核苷酸交换因子和 GAP 存在的情况下，将测量初级和次级同位素效应，以确定 Ras、Ran 和 Galpha-i1 催化的 GTP 水解过渡态的性质。应进行由含有活性位点突变的 Galpha-i1 催化的 GTPase 反应的 KIE，以探讨催化残基在过渡态形成中的作用。与同位素比质谱仪耦合的灵敏化学反应接口可用于精确确定远程 (13)C 标记的 (18)O-GTP 底物和产物的相对同位素取代。所提出的方法仅需要纳摩尔量的同位素标记底物，因此适用于具有重要生化和生理意义的各种 ATP 酶和 GTP 酶的机制研究。