Methods for Determining Glycosyltransferase Kinetics
Glycosyltransferases are a class of biosynthetic enzymes that transfer individual activated monosaccharide units to specific acceptors. Colorimetric assays using the detection of released products such as para-nitrophenol and coupled assays for inorganic phosphate detection allow for convenient and quantifiable kinetic characterization. These techniques may be applied to determine the enzymatic activity of glycosyltransferases by indirectly measuring the transfer of nucleotide-activated donor carbohydrate units to various cognate acceptor molecules. In addition to an overview of these methods, the protocol for quantifying the glycosyltransferase activity used for the characterization of penicillin-binding proteins (PBPs) involving the transfer of lipid II to form elongated murein chains during bacterial cell wall synthesis is described herein.
Key wordsGlycosyltransferases Enzyme kinetics Malachite green dye para-Nitrophenol Colorimetric assay Lipid II transfer
This work was supported by NSERC grant RGPIN-2014-05018 and the Faculty of Science, Wilfrid Laurier University.
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