Enzymatic transglycosylation by the Ping Pong bi bi mechanism: Selectivity for transglycosylation versus primary and secondary hydrolysis

There has been a recent surge of interest in producing oligosaccharides by transglycosylation. In enzyme-catalyzed transglycosylations that follow the Ping Pong bi bi mechanism, there are three competing reactions: the transglycosylation itself, primary hydrolysis (i.e. of the initial donor molecule) and secondary hydrolysis (i.e. of the transglycosylation product). In kinetically controlled transglycosylations, the maximum yield of the transglycosylation product depends on the selectivities of the enzyme for transglycosylation in relation to these hydrolysis reactions. Current methods for determining selectivity only characterize the selectivity for transglycosylation in relation to primary hydrolysis. In the current work, we develop a model that can be used to estimate both selectivities from a reaction profile of percentage yield of transglycosylation product plotted against the percentage consumption of the donor molecule. We use case studies from the literature to show how the model can be used to quantify the results of mutations aimed at improving selectivity. We also use the model to explore strategies for improving the yield of the transglycosylation product. This exploration demonstrates that it is not sufficient to have high selectivities for transglycosylation in relation to hydrolysis, it is also necessary that the transglycosylation reaction have a reasonably high equilibrium constant.