<p>MurA catalyzes the first committed step of peptidoglycan biosynthesis and it is the target of the antibiotic fosfomycin. Due to a Cys-to-Asp substitution in the active site, MurAs from a number of pathogenic bacteria, including <em>Mycobacterium tuberculosis</em> and <em>Borrelia burgdorferi</em> (Lyme disease), are fosfomycin resistant. His-tagged <em>Borrelia burgdorferi</em> MurA (Bb_MurA) and its D116C mutant have been successfully expressed, purified and characterized. The <em>k</em>_cat value of wild-type Bb_MurA was 0.74 ± 0.01 s^-1. The D116C mutant’s <em>k</em>_cat decreased by 25-fold and was fosfomycin sensitive. The pH profiles of <em>k</em>_cat for both Bb_MurA and its mutant were characterized. There was little difference in p<em>K</em>_a1 values, but the p<em>K</em>_a2 value shifted from 7.4 ± 0.2 in wild-type enzyme to a value >11 in the mutant. This demonstrated that the p<em>K</em>_a2 of 7.4 was due to D116, and that it must be protonated for activity. Fosfomycin inactivation of Bb_MurA_H6(D116C) was time-dependent and only proceeded in the presence of UDP-GlcNAc. The dissociation constant, <em>K</em>_i, was 5.7 ± 0.4 µM and rate of covalent modification, <em>k</em>_inact, was 0.021 ± 0.003 s^-1.</p> <p>DAHP synthase catalyzes the first committed step in the shikimate pathway, and its catalysis has been proposed to proceed through two oxacarbenium ion intermediates. Pyruvate oxime, glyoxylate oxime and 4-imidazolecarboxylic acid have been evaluated as inhibitors of DAHP synthase. In the presence of glycerol 3-phosphate, the fitted <em>K</em>_i values of pyruvate oxime and glyoxylate oxime were 7.6 (± 0.9) × 10^-5 M and 7.4 (± 1.7) × 10^-5 M, respectively. 4-Imidazolecarboxylic acid’s inhibition was cooperative, and its binding was competitive with respect to PEP, and uncompetitive with respect to E4P. Its equilibrium dissociation constant was 3.0 (± 0.2) × 10^-3 M.</p> / Master of Science (MSc)

The research work reported in the thesis deals with the development of dynamic covalent systems and their applications in evaluation and screening of protein-ligands and enzyme inhibitors, as well as in synthetic methodologies. The thesis is divided into four parts as described below. In part one, synthetic methodologies to access 3-functionalized phthalides and 3-thioisoindolinones using the concept of cascade reactions are demonstrated. Efficient syntheses of the target products are designed and performed in one-pot process under mild reaction conditions.  In part two, phosphine-catalyzed disulfide metathesis for the generation of dynamic carbohydrate system in aqueous solution is demonstrated. In the presence of biological target (Concanavalin A), the optimal dynamic ligand is successfully identified in situ by the 1H STD-NMR spectroscopy. In part three, lipase-catalyzed resolutions of dynamic reversible systems using reversible cyanohydrin and hemithioacetal reactions in one-pot processes are demonstrated. The dynamic systems are generated under thermodynamic control in organic solution and subsequently resolved by lipase-mediated resolution under kinetic control. The resolution processes resulted in the lipase-selected substrates with high structural and stereochemical specificities. In the last part, dynamic fragment-based strategy is presented using β-galactosidase as a model target enzyme. Based on our previous study, the best dynamic inhibitor of β-galactosidase was identified using 1H STD-NMR technique from dynamic hemithioacetal systems. The structure of the dynamic inhibitor is tailored by fragment linking and optimization processes. The designed inhibitor structures are then synthesized and tested for inhibition activities against β-galactosidase. / QC 20110526