<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><sub>cat</sub> value of wild-type Bb_MurA was 0.74 ± 0.01 s<sup>-1</sup>. The D116C mutant’s <em>k</em><sub>cat</sub> decreased by 25-fold and was fosfomycin sensitive. The pH profiles of <em>k</em><sub>cat</sub> for both Bb_MurA and its mutant were characterized. There was little difference in p<em>K</em><sub>a1</sub> values, but the p<em>K</em><sub>a2</sub> 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><sub>a2</sub> of 7.4 was due to D116, and that it must be protonated for activity. Fosfomycin inactivation of Bb_MurA<sub>H6</sub>(D116C) was time-dependent and only proceeded in the presence of UDP-GlcNAc. The dissociation constant, <em>K</em><sub>i</sub>, was 5.7 ± 0.4 µM and rate of covalent modification, <em>k</em><sub>inact</sub>, was 0.021 ± 0.003 s<sup>-1</sup>.</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><sub>i</sub> values of pyruvate oxime and glyoxylate oxime were 7.6 (± 0.9) × 10<sup>-5</sup> M and 7.4 (± 1.7) × 10<sup>-5</sup> 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<sup>-3</sup> M.</p> / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/10718 |
| Date | 10 1900 |
| Creators | Jiang, Shan |
| Contributors | Berti, Paul, Giuseppe Melacini, Murray Junop, Giuseppe Melacini, Murray Junop, Chemical Biology |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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