Function determination and kinetics of NagS.

<p>(<b>a</b>) <sup>1</sup>H NMR spectrum of the enzymatic reaction mixture of GlcNAc-6P with either the active NagS (top) or the heated-inactivated one (bottom). The associated NMR peaks of the reaction product are highlighted in red circles and arrows. (<b>b</...

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Hlavní autor: Chao Li (145513) (author)
Další autoři: Mia Urem (22683580) (author), Ioli Kotsogianni (9930187) (author), Josephine Lau (20442343) (author), Chao Du (288036) (author), Somayah S. Elsayed (9192571) (author), Nathaniel I. Martin (847378) (author), Iain W. McNae (840777) (author), Patrick Voskamp (2311429) (author), Christoph Mayer (57204) (author), Sébastien Rigali (724082) (author), Navraj Pannu (10163763) (author), Jan Pieter Abrahams (1429531) (author), Lennart Schada von Borzyskowski (22683583) (author), Gilles P. van Wezel (7838948) (author)
Vydáno: 2025
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Shrnutí:<p>(<b>a</b>) <sup>1</sup>H NMR spectrum of the enzymatic reaction mixture of GlcNAc-6P with either the active NagS (top) or the heated-inactivated one (bottom). The associated NMR peaks of the reaction product are highlighted in red circles and arrows. (<b>b</b>) HRESIMS spectrum of the NagS product, compound <b>1</b>: <i>m</i>/<i>z</i> 282.0387 [M-H]<sup>−</sup> (calculated for C<sub>8</sub>H<sub>13</sub>NO<sub>8</sub>P, 282.0384) <b>(c)</b> <sup>1</sup>H NMR spectrum of the enzymatic reaction mixture of ManNAc-6P with either the active NagS (top) or the heat-inactivated one (bottom). The associated NMR peaks of the reaction product are highlighted in red circles. (<b>d</b>) Reactions catalyzed by NagS. NagS dehydrates both GlcNAc-6P and ManNAc-6P to produce compound <b>1</b>. Michaelis-Menten curves were fitted, and selected curves are shown for NagS with the substrates GlcNAc-6P (<b>e</b>) with <i>K</i><sub>m</sub> value of 0.45 ± 0.03 mM and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> value of 5.48 × 10<sup>4</sup> M<sup>−1</sup>·s<sup>−1</sup>, and ManNAc-6P (<b>f</b>) with <i>K</i><sub>m</sub> value of 0.68 ± 0.05 mM and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> value of 1.32 × 10<sup>3</sup> M<sup>−1</sup>·s<sup>−1</sup>. In <b>e</b> and <b>f</b>, the <i>V</i><sub>0</sub> data were plotted against the substrate concentration, and each assay was performed in triplicate and expressed as a mean ± standard error. The data underlying this Figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3003514#pbio.3003514.s022" target="_blank">S1 Data</a>.</p> <p>(TIF)</p>