Characterization of sialyltransferase mutants using surface plasmon resonance

Sialyltransferases are enzymes responsible for the important sialylation of glycoconjugates. Since crystal structures are not available, other tools a re needed to study enzymatic mechanisms. As a model, we used human alpha2,6 -sialyltransferase. A putative acceptor-binding domain containing the small and the very small sialyl motifs was randomly mutated. This resulted in en zymes with altered enzymatic activity. Affinity chromatography demonstrated that their binding to donor substrate was maintained. To illustrate the ro le of the mutated domain in acceptor binding, a method based on surface pla smon resonance was set up. Only at low salt and high acceptor concentration was association of wildtype ST6GalI with asialofetuin demonstrated. As exp ected, this interaction was affected by cytidine 5'-monophospho-N-acetylneu raminic acid, the donor substrate, which proves the specificity of the inte raction. Different types of mutants were found. For some, the drop in activ ity could be explained by loss in affinity for the acceptor. For others, th e catalytic center, but not the acceptor-binding site, was affected. Neithe r acceptor binding nor catalytic activity were limited to the sialyl motifs . To our knowledge, this is the first example in which surface plasmon reso nance is successfully used to demonstrate the binding of a glycosyltransfer ase to its natural acceptor.