Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry |
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Authors: | Florence Gaucher-Wieczorek,Vincent Gué rineauDavid Touboul,Sophie Thé tiot-LaurentFranck Pelissier,Marie-Ange Badet-DenisotBernard Badet,Philippe Durand |
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Affiliation: | Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France |
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Abstract: | Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5′-diphospho-N-acetyl-d-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts d-fructose-6-phosphate (Fru-6P) and l-glutamine (Gln) into d-glucosamine-6-phosphate (GlcN-6P) and l-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. |
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Keywords: | Glucosamine-6P synthase MALDI-TOF Enzyme assay Bisubstrate enzyme |
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