title: An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase.
abstract: An assay for glucosamine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) as coupling enzyme was developed. GNA1 transfers the acetyl moiety from acetyl-coenzyme A (CoA) to glucosamine-6-phosphate, releasing coenzyme A. The assay measures the production of glucosamine-6-phosphate by either following the consumption of acetyl-CoA spectrophotometrically at 230nm or quantifying the free thiol with 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) in a discontinuous manner. This method is simple to perform and can be adapted to a 96-well microtiter plate format, which will facilitate high-throughput inhibitor screening and mechanistic studies using purified GlmS.
authors: Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-Sur-Yvette, France.
authors: Yanyan
authors: Y
authors: Li
authors: Philippe
authors: P
authors: Lopez
authors: Philippe
authors: P
authors: Durand
authors: Jamal
authors: J
authors: Ouazzani
authors: Bernard
authors: B
authors: Badet
authors: Marie-Ange
authors: MA
authors: Badet-Denisot
mesh_terms: D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000378:metabolism*; D007700:Kinetics; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D000637:Transaminases / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D000637:Transaminases; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)