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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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> |
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