(18)O Kinetic isotope effects in G protein GTPases

描述（由申请人提供）：拟议研究的目标是确定G蛋白催化GTP水解的过渡态和机制。G蛋白，包括Ras超家族成员和异源三聚体G蛋白（G α）的α亚基，当与GTP结合时是信号转导子，但因其内在或GTP酶活性而失活。在体内，GT3活化蛋白可加速GT3活性。固有的GT3活性是缓慢的，催化位点的构象变化可能是限速的。GAP蛋白通过降低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固有的GTTR速率。小G蛋白Ras和Ran以及异源三聚体G α蛋白的GAP似乎通过不同的机制加速其G蛋白底物的GTdR活性。特别感兴趣的是GAP蛋白是否改变G蛋白催化的GTdR反应的过渡态。建议测量特异性（18）O-标记的GTP底物水解的动力学同位素效应（KIE），以确定构象或化学步骤是否是G蛋白和GAP促进的GTP水解的速率限制。在适当的核苷酸交换因子和GAP存在下，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水解的过渡态性质。应进行含有活性位点突变的Galpha-i1催化的GTdR反应的KIE，以探测催化残基在过渡态形成中的作用。应使用与同位素比质谱仪耦合的灵敏化学反应界面，精确测定远程（13）C标记（18）O-GTP底物和产物的相对同位素取代。所提出的方法只需要纳摩尔量的同位素标记的底物，因此适用于各种ATP酶和GTP酶的重要的生物化学和生理学利益的机制研究。

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.