Spelling suggestions: "subject:"enolpyruvylshikimate."" "subject:"phosphoenolpyruvat.""
1 |
Design and synthesis of potential inhibitors of enolpyruvyl shikimate 3-phosphate synthase (EPSPS)Gawuga, Vivian 10 1900 (has links)
The emergence of antibiotic resistance to current treatments of bacterial
infection represents a major challenge that needs to be addressed with the
development of new generations of inhibitors. The enzyme 5-enolpyruvylshikimate 3-
phosphate synthase (EPSPS) catalyses the sixth step in the shikimate biosynthetic
pathway, which is essential for the synthesis of aromatic compounds such as the
aromatic amino acids phenylalanine, tryptophan and tyrosine. It occurs in plants,
bacteria and some parasites. Since the pathway is absent in mammals but essential for
the pathogenicity of a number of organisms, EPSPS is considered a prospective target
for new inhibiter design. A number of EPSPS inhibitors have been reported in the
literature. What we are lacking is an understanding of the features that are important
for binding EPSPS. We have synthesized compounds to probe the active site of the
enzyme based on the knowledge of an enzyme-catalyzed intermediate with a high
cationic character. This will include assembling bipartite/tripartite inhibitors to
discover what interactions or structural motifs are important for binding. Once the
features important for binding to EPSPS are understood, the possibility of elaborating
them to create potent inhibitors of EPSPS will be investigated. In addition, the
synthesis of two shikimate analogs [5-^(18)O] shikimic acid and 4-deoxyshikimic acid
were completed for further experiments to probe the enzyme mechanism in detail, and for transition state structure by transition state analysis. Transition state analysis using
kinetic isotopic effects (KIE) will elucidate the transition state structure of the
enzyme-catalyzed EPSP reaction, and provide a detailed starting point for designing
EPSPS inhibitors. / Thesis / Master of Science (MSc)
|
2 |
Understanding the AroA Mechanism: Evidence for Enolpyruvyl Activation and Kinetic Isotope Effect MeasurementsClark, Meghann E. 08 1900 (has links)
<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)
|
Page generated in 0.3612 seconds