AroA catalyzes the sixth step of the shikimate biosynthetic pathway which produces aromatiG amino acids in plants and bacteria, but is absent in mammals. This makes AroA an attractive antimicrobial target. The transition state (TS) structures of AroA- and acid-catalyzed 5-eno/pyruvyl shikimate-3-phosphate (EPSP) hydrolysis were studied in atomic detail by kinetic isotope effect (KIE) measurement. Enzymes bind their transition states more tightly than any other species, so molecules that closely resemble the transition state would have a high affinity for the enzyme and be good inhibitors. Radiolabelled EPSPs were synthesized and a KIE measurement method was developed. Six KIEs were measured for both the AroA- and acid-catalyzed reactions. KIEs for the AroA reaction indicate a cationic TS structure. The acid-catalyzed reaction may employ a slightly different mechanism with an earlier TS. A computational TS model was found and its KIEs were calculated. It demonstrated good agreement with the experimental values at most positions. The model is being modified to improve the agreement with the experimental KIEs. This TS structure will be a good starting point for inhibitor design. All these efforts, hopefully, can make a positive contribution to the development of antimicrobial drugs. / Thesis / Master of Science (MSc)

<p> AroA catalyzes a carboxyvinyl transfer reaction, forming enolpyruvyl shikimate 3-phosphate (EPSP) from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP). Upon extended incubation, it forms EPSP ketal by intramolecular nucleophilic attack of O4H on C2' of the enolpyruvyl group. EPSP ketal was previously proposed to form by non-enzymatic breakdown of the tetrahedral intermediate (THI) which had dissociated from AroA. In this study, EPSP ketal formed in the presence of excess AroA, which demonstrated that it was formed in the active site. This eliminated non-enzymatic THI breakdown as its source, and demonstrated that AroA forms either a discrete EPSP cationic intermediate, or cl transition state with high cationic character. The pH dependence of non-enzymatic EPSP hydrolysis was examined in order to understand the intrinsic reactivity of the enolpyruvyl group. Acid catalysis accelerated EPSP hydrolysis> 10^8-fold. These results provide evidence for enolpyruvyl activation through protonation at C3', forming an unstable cationic intermediate, or a highly cation-like transition state. The incorporation of 2H into EPSP from solvent 2H20 during AreA-catalyzed hydrolysis was much slower than the hydrolysis rate, in the absence of inorganic phosphate in the reaction. This demonstrated that KIEs on AroA-catalyzed EPSP hydrolysis, when they are measured in the future, will reflect protonation of EPSP. A method was developed for KIE measurements on acid-catalyzed EPSP hydrolysis, which showed good reproducibility and no buffer dependence. Further experiments need to be completed on the acid-catalyzed KIEs and enzyme-catalyzed KIEs, followed by transition state analysis. This will precisely define the transition state structure of the enzyme-catalyzed EPSP hydrolysis reaction, and provide a good starting point for designing AroA inhibitors.</p> / Thesis / Master of Science (MSc)