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Anal Biochem. 2007 Nov 15;370(2):142-6. Epub 2007 Aug 9.

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

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1
Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-Sur-Yvette, France.

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.

PMID:
17880906
DOI:
10.1016/j.ab.2007.07.031
[Indexed for MEDLINE]
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