Investigation of Substrate Specificity, Mechanism, and Inhibition of IGPS

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Project Summary / Abstract IGP Synthase (IGPS) is a promising new antibiotic drug target. This enzyme plays a critical role in tryptophan biosynthesis in bacteria but is not present in humans. The overall goals of this proposal are to gain new understanding of various substrate-IGPS interactions and to develop new IGPS inhibitors which may have potential as anti-infective agents. Thus, this work is highly relevant to understanding and treating intractable and widespread resistant bacteria. We will use IGPS from M. tuberculosis (MtIGPS) as a model system. New MtIPGS substrate analogs will be strategically designed in such a way that their behavior with respect to the enzyme will answer questions about IGPS structure-function relationships and catalysis. A series of new substrate analogs with altered or removed functional groups and aromatic ring substitutions will be synthesized. The analogs' ability to bind and act as substrates for MtIGPS will be determined. These data will be systematically compared to those obtained with the natural substrate and an unreactive reduced substrate analog. These experiments will determine the role and importance of the substrate functional groups and reveal which ones must be preserved and which ones can be modified for inhibitor optimization. Several MtIGPS active site residues suspected to play a role in ligand binding will be replaced using mutagenesis. The effects of these mutations on kinetic parameters will provide insights into the role of each residue in binding and catalysis. These data will reveal the importance of specific intermolecular interactions between IGPS ligands and the active site, and they complement the data obtained with substrate analogs. The generally-accepted mechanism for IGPS catalysis begins with an endothermic intramolecular cyclization and de-aromatization of the substrate. MtIGPS likely facilitates this step by binding strongly or conformationally collapsing around the resulting intermediate or a structurally-similar preceding transition state. New MtIGPS inhibitors designed to be converted enzymatically into transition state analogs without turning over to form products will be synthesized. The inhibitory properties and the effects of these new compounds on the IGPS kinetic pathway will be determined.