Kinetic evidence of the existence of a stable enzyme-glycosyl intermediary complex in the reaction catalyzed by endotransglycosylase.

Xyloglucan-endotransglycosylase (XET) is an enzyme involved in the metabolism of xyloglucan (XG) in plant cell walls and seeds. This enzyme acts both as a hydrolase and as a transglycosylase by transferring the fragments of xyloglucan molecules to other XG molecules or xyloglucan-derived oligosaccharides (XGOs). In this work, we studied the kinetics of interaction between XET and XG. The equilibrium in the reaction of XG degradation by XET was found to depend on the initial enzyme concentration and the availability of suitable glycosyl acceptors. After reaching the equilibrium, the addition to the reaction mixture of XET or XGOs caused further degradation of XG, and a new equilibrium with a higher degree of XG depolymerization was established. These results indicated that in the course of XG depolymerization, the enzyme is bound in a relatively stable, temporarily inactive enzyme-glycosyl complex and this complex is decomposed by transferring its glycosyl moiety to suitable oligosaccharide acceptor. Mouse polyclonal antibody against XET linked to AffiGel 10 (Affi-Ab) adsorbed both XET and XET-XG complex but not [3H]XG alone. XET immobilized onto Affi-Ab was able to bind [3H]XG and catalyze transglycosylation in presence of XGOs.