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DC Field | Value | Language |
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dc.contributor.advisor | Berti, Paul | en_US |
dc.contributor.author | Popović, Vladimir | en_US |
dc.date.accessioned | 2014-06-18T16:57:23Z | - |
dc.date.available | 2014-06-18T16:57:23Z | - |
dc.date.created | 2012-02-02 | en_US |
dc.date.issued | 2012-04 | en_US |
dc.identifier.other | opendissertations/6820 | en_US |
dc.identifier.other | 7841 | en_US |
dc.identifier.other | 2490676 | en_US |
dc.identifier.uri | http://hdl.handle.net/11375/11888 | - |
dc.description.abstract | <p>Sialic acid synthase (NeuB) is a key enzyme in bacterial biosynthesis of the sialic acid <em>N</em>-acetylneuraminic acid (NeuNAc). It catalyzes the addition of phosphoenolpyruvate (PEP) to <em>N</em>-acetylmannosamine (ManNAc) in the presence of a divalent cation such as Mn<sup>2+</sup>. We have explored the inhibition of NeuB by an oxacarbenium ion mimic, NeuNAc oxime, and hydroxylamine (NH<sub>2</sub>OH). NeuNAc oxime shows slow-binding inhibition with a binding half-life of 2.5 h and an inhibition constant (<em>K</em><sub>i</sub><sup>*</sup>) of 1.6(± 0.7) pM. Even though NeuNAc oxime binds NeuB with high affinity, there remains approximately 10% residual activity even after extended pre-incubation with high inhibitor concentrations. In contrast, in the presence of substrates, when NeuB was actively catalyzing NeuNAc synthesis, complete inhibition by NeuNAc oxime was observed within 6 h. This inhibition profile is similar to NH<sub>2</sub>OH; which has previously been shown to elicit complete, time-dependent inhibition. We propose the existence of two NeuB conformations: an asymmetric idle state conformation (NeuB<sup>IS</sup>), in which NeuNAc oxime is able to bind to only one monomer of this dimeric enzyme, and a second conformation, running state NeuB (NeuB<sup>RS</sup>), which is completely inhibited due to either NeuNAc oxime binding to the second monomer, or the dimer adopting a conformation in which the unbound monomer is inactive. Experiments with [1-<sup>14</sup>C]PEP showed that in the presence of large excess of substrate, inhibition occurred faster than with a lower excess. This suggests that a sustained buildup of NeuB<sup>RS<strong> </strong></sup>is required for complete inhibition.</p> | en_US |
dc.subject | sialic acid | en_US |
dc.subject | NeuB | en_US |
dc.subject | oxime | en_US |
dc.subject | oxacarbenium ion | en_US |
dc.subject | enzyme inhibition | en_US |
dc.subject | slow-binding inhibitor | en_US |
dc.subject | Neiserria meningitidis | en_US |
dc.subject | residual activity | en_US |
dc.subject | NeuNAc | en_US |
dc.subject | siaC | en_US |
dc.subject | Neu5Ac | en_US |
dc.subject | NANA | en_US |
dc.subject | Biochemistry | en_US |
dc.subject | Medicinal Chemistry and Pharmaceutics | en_US |
dc.subject | Pharmacology | en_US |
dc.subject | Biochemistry | en_US |
dc.title | Inhibition of the bacterial sialic acid synthase, NeuB | en_US |
dc.type | thesis | en_US |
dc.contributor.department | Chemical Biology | en_US |
dc.description.degree | Master of Science (MSc) | en_US |
Appears in Collections: | Open Access Dissertations and Theses |
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File | Size | Format | |
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fulltext.pdf | 4.45 MB | Adobe PDF | View/Open |
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