INVESTIGATION OF SUBSTRATE SPECIFICITY, MECHANISM, AND INHIBITION OF IGPS

Project Summary / Abstract IGP Synthase (IGPS) is a promising new antibiotic drug target. This enzyme plays a critical rolein tryptophan biosynthesis in bacteria but is not present in humans. The overall goals of this proposalare to gain new understanding of various substrate-IGPS interactions and to develop new IGPS inhibitorswhich may have potential as anti-infective agents. Thus, this work is highly relevant to understanding andtreating intractable and widespread resistant bacteria. We will use IGPS from M. tuberculosis (MtIGPS) as a model system. New MtIPGS substrateanalogs will be strategically designed in such a way that their behavior with respect to the enzyme willanswer questions about IGPS structure-function relationships and catalysis. A series of new substrateanalogs 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 besystematically compared to those obtained with the natural substrate and an unreactive reducedsubstrate analog. These experiments will determine the role and importance of the substrate functionalgroups and reveal which ones must be preserved and which ones can be modified for inhibitoroptimization. Several MtIGPS active site residues suspected to play a role in ligand binding will be replacedusing mutagenesis. The effects of these mutations on kinetic parameters will provide insights into therole of each residue in binding and catalysis. These data will reveal the importance of specificintermolecular interactions between IGPS ligands and the active site, and they complement the dataobtained with substrate analogs. The generally-accepted mechanism for IGPS catalysis begins with an endothermic intramolecularcyclization and de-aromatization of the substrate. MtIGPS likely facilitates this step by binding strongly orconformationally collapsing around the resulting intermediate or a structurally-similar preceding transitionstate. New MtIGPS inhibitors designed to be converted enzymatically into transition state analogs withoutturning over to form products will be synthesized. The inhibitory properties and the effects of these newcompounds on the IGPS kinetic pathway will be determined.>