(18)O Kinetic isotope effects in G protein GTPases

描述(申请人提供)：拟议研究的目标是确定G蛋白催化的GTP水解酶的过渡状态和机制。G蛋白包括RAS超家族成员和异三聚体G蛋白(Galpha)的α亚基，当与GTP结合时是信号转导，但被其固有的或GTPase活性灭活。在体内，GTP酶激活蛋白可以促进GTP酶的活性。固有的GTP酶活性缓慢，催化部位的构象变化可能是限速的。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酶的速率。小G蛋白Ras和Ran以及异三聚体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。尤其令人感兴趣的是，GAP蛋白是否会改变G蛋白催化的GTP酶反应的过渡态。有人建议测量特定的(18)O标记GTP底物的动力学同位素效应(Kie)，以确定构象步骤或化学步骤对G蛋白和GAP促进的GTP水解是速率限制的。在适当的核苷酸交换因子和间隙存在的情况下，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。为了探索催化残基在过渡态形成中的作用，应对含有活性位点突变的Galpha-i1催化的GTP酶反应进行KIE。应使用与同位素比质谱仪连接的灵敏化学反应界面来精确测定远程(13)C标记(18)O-GTP底物和产物的相对同位素取代度。所提出的方法只需要纳摩尔量的同位素标记的底物，因此适用于各种具有重要生化和生理意义的ATPase和GTP酶的机械研究。

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.