ALLOSTERIC MECHANISMS OF SYNCHRONIZATION WITHN THE TRIAD GLUTAMIINE ADMIDOTRANSF

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 谷氨酰胺氨基转移酶家族(GATS)具有催化谷氨酰胺在谷氨酰胺酶(GATase)结构域中的水解酶活性，并将生成的氨水通过溶剂无法进入的通道转移到远距离(10-40A)氨基低聚酶(ALase)结构域中的受体-底物的能力。这两个活性位点的同步被认为涉及GATase活性的变构激活，通过受体-底物、过渡态和/或变构效应物的ALASE结构域内的关联。本课题组试图通过研究模型酶CTP合成酶(CTPS)的GATase结构域诱导的构象变化来阐明同步化的机制。CTPs定义了细胞内CTP的水平，因此是一个重要的抗肿瘤靶点，并通过变构效应GTP与其核苷酸底物的结合激活其GATase活性。我们的方法是使用定点突变在CPT的区域内引入色氨酸探针，这些区域被认为与促进GATase活性的变构信号密切相关。每个探针的动态荧光性质将对到目前为止结构研究中尚未解决的微妙扰动敏感和描述，对底物、产物和变构效应器的所有可能组合的系统评估将提供有助于协调信号刺激的特定分子组合的细节。在这项为期一年的资助下，我们特别寻求：(1)解决配体诱导的CTPS天然色氨酸的荧光变化的复杂基质，从而直接洞察UTP、ATP和GTP激活GATase活性的变构机制；以及(2)测试CTPS中的同步性源于GATase结构域环结构的动态变化，该结构优化了“氧阴离子空穴”的形成。最终，了解决定该生物合成酶家族内部变构协调的结构/功能关系将有助于在其控制的基础上进一步开发抗癌、抗病毒和抗寄生虫药物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Members of the Triad Glutamine Amidotransferase Family (GATs) share the ability to catalyze the hydrolyssi of glutamine within a glutaminase (GATase) domain and transfer the resulting ammonia through a solvent-inaccessible tunnel to an acceptor-substrate within a distant (10-40A) amidoligase (ALase) domain. Synchronization of the two active sites is thought to involve the allosteric activation of GATase activity via association within the ALase domain of acceptor-substrates, transition states, and/or allosteric effectors. Our group seeks to elucidate mechanisms of synchronization by examining the conformational changes induced withini the GATase domain of the model enzyme CTP synthetase (CTPS). CTPS defines intracellular levels of CTP, is consequently an important antineoplastic target, and is activated in its GATase activity via the binding of the allosteric effetor GTP and its nucleotide substrates. Our approach is to use site-directed mutagenesis to introduce tryptophan probes within regions of CPTS proposed to be intimate to the allosteric signal promoting GATase activity. The dynamic, fluorescence properties of each probe will be sensitive to and descriptive of the subtle perturbations that have thus far eluded structural studies, and a systmatic evaluation of all possible combinations of substrates, products, and allosteric effectors will provide details on the specific combination of molecules contributing to the stimulation of the coordinating signal. Under the auspices of this one-year grant, we specifically seek: (1) to resolve the complex matrix of ligand-induced, fluorescence changes in the native tryptophans of CTPS, thus providing direct insight into allosteric mechanisms directing the activation of GATase activity by UTP, ATP and GTP; and (2) to test the hypothesis that synchronization in CTPS derives from alterations in the dynamics of GATase domain loop structures that optimize "oxyanion hole" formation. Ultimately, knowledge of the structure/function relationships dictating allosteric coordination within this family of biosynthetic enzymes will facilitate the further development of anti-cancer, anti-viral, and anti-parasitic pharmaceuticals based on their control.