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.
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.
Maillard
Ludovic T
LT
Institut de Chimie des Substances Naturelles, UPR 2301-CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Guérineau
Vincent
V
Badet-Denisot
Marie-Ange
MA
Badet
Bernard
B
Laprévote
Olivier
O
Durand
Philippe
P
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
D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine