Please use this identifier to cite or link to this item:
http://hdl.handle.net/11375/11888
Title: | Inhibition of the bacterial sialic acid synthase, NeuB |
Authors: | Popović, Vladimir |
Advisor: | Berti, Paul |
Department: | Chemical Biology |
Keywords: | sialic acid;NeuB;oxime;oxacarbenium ion;enzyme inhibition;slow-binding inhibitor;Neiserria meningitidis;residual activity;NeuNAc;siaC;Neu5Ac;NANA;Biochemistry;Medicinal Chemistry and Pharmaceutics;Pharmacology;Biochemistry |
Publication Date: | Apr-2012 |
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> |
URI: | http://hdl.handle.net/11375/11888 |
Identifier: | opendissertations/6820 7841 2490676 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Size | Format | |
---|---|---|---|
fulltext.pdf | 4.45 MB | Adobe PDF | View/Open |
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.