title: Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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.
authors.affiliation: Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Gaucher-Wieczorek
authors.affiliation: Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Guérineau
authors.affiliation: Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. Electronic address: david.touboul@cnrs.fr.
authors.forename: David
authors.initials: D
authors.lastname: Touboul
authors.affiliation: Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Thétiot-Laurent
authors.affiliation: Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Franck
authors.initials: F
authors.lastname: Pelissier
authors.affiliation: Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Marie-Ange
authors.initials: MA
authors.lastname: Badet-Denisot
authors.affiliation: Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Badet
authors.affiliation: Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Durand
mesh_terms: D057075:Enzyme Assays; D005636:Fructosephosphates / Q000032:analysis / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism; D005973:Glutamine / Q000032:analysis / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000378:metabolism*; D007700:Kinetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D005636:Fructosephosphates; D005973:Glutamine; C001293:glucosamine 6-phosphate; D018698:Glutamic Acid; D019298:Glucose-6-Phosphate; C027618:fructose-6-phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine
keywords: Bisubstrate enzyme; Enzyme assay; Glucosamine-6P synthase; MALDI-TOF