An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase.
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.
Li
Yanyan
Y
Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-Sur-Yvette, France.
Lopez
Philippe
P
Durand
Philippe
P
Ouazzani
Jamal
J
Badet
Bernard
B
Badet-Denisot
Marie-Ange
MA
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*
D029701:Saccharomyces cerevisiae Proteins; D000637:Transaminases; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)