title: Monitoring enzyme-catalyzed production of glucosamine-6P by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new enzymatic assay for glucosamine-6P synthase.
abstract: A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) method for quantification of D-glucosamine-6P (GlcN-6P) that allows the kinetic study of glucosamine-6P synthase (Glms) is presented. The present report describes the optimization of the different steps of a new enzymatic assay for Glms based on in situ N-acetylation of GlcN-6P and MALDI-TOFMS analysis using N-(13C2)acetylglucosamine-6P as internal standard. Since no isotopically substituted GlcN-6P was available, the N-(13C2)acetyl derivative, easily obtained from (13C4)-acetic anhydride, was used as internal standard. Validation of the assay was achieved by measuring the fructose-6P Michaelis constant, in full agreement with reported values, and by studying the inhibition properties of arabinose-5P oxime.
authors.affiliation: Institut de Chimie des Substances Naturelles, UPR 2301-CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
authors.forename: Ludovic T
authors.initials: LT
authors.lastname: Maillard
authors.forename: Vincent
authors.initials: V
authors.lastname: Guérineau
authors.forename: Marie-Ange
authors.initials: MA
authors.lastname: Badet-Denisot
authors.forename: Bernard
authors.initials: B
authors.lastname: Badet
authors.forename: Olivier
authors.initials: O
authors.lastname: Laprévote
authors.forename: Philippe
authors.initials: P
authors.lastname: Durand
mesh_terms: D002138:Calibration; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D005944:Glucosamine / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000032:analysis* / Q000378:metabolism*; D015394:Molecular Structure; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine