An enzyme-coupled assay for amidotransferase activity of glucosamine-6-phosphate synthase

An assay for gluco samine-6-phosphate synthase using a yeast glucosamine-6-phosphate N-acetyltransferase l (GNAl) as coupling enzyme was developed. GNAl 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 230 nm 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 GImS. (c) 2007 Elsevier Inc. All rights reserved.