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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Exploratory Grants (P20)
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Special Emphasis Panel (ZRR1-RI-4 (01))
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University of Oklahoma Health Sciences Center
Schools of Medicine
Oklahoma City
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