Glycans are important molecules and/or components of larger entities (like glycoproteins). Glycosyltransferases achieve the biosynthesis of glycans by catalyzing the specific transfer of carbohydrate moieties from an activated donor to a specific acceptor substrate. Human fucosyltransferase 5 catalyzes the transfer of a fucose residue from GDP-Fucose to form the Lewis X Antigen. This antigen is recognized by several human lectins. This antigen is known to play an important role in the metastasis of certain cancer types, as the tumor tissue overexpresses the fucosyltransferase and consequently also the Lewis X antigen. Thereby tumor cells can adhere to endothelial lectins and relocate to healthy tissue to produce metastases. Knowledge of the underlying reaction mechanisms is the basis to produce new inhibitors and potentially stop cancer progression.  Here we show for the first time the real time kinetics of this important enzyme measured by NMR. Both acceptor and donor Km and kcat can be measured in a single experiment using the integrated form of the Michaelis Menten kinetics. Side products, as the autohydrolysis of the donor, are traced and can easily be quantified. Metal ion dependency as well as the course of the enzymatic reaction will be discussed.

Real time kinetics of human fucosyltransferase 5

247th National Meeting (2014)

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