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 met abolism of xyloglucan (XG) in plant, cell walls and seeds. This enzyme acts both as a hydrolase and as a transglycosylase by transfering the fragments of xyloglucan molecules to other XG molecules or xyloglucan -derived oligosaccharides (XGOs). In this work, we studied the kinetic s of interaction between XET and XG. The equilibrium in the reaction o f XG degradation by XET was found to depend on the initial enzyme conc entration and the availability of suitable glycosyl accepters. After r eaching the equilibrium, the addition to the reaction mixture of XET o r XGOs caused further degradation of XG, and a new equilibrium with a higher degree of XG depolymerization was established. These results in dicated 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 transfering its glycosyl moiety to s uitable oligosaccharide acceptor. Mouse polyclonal antibody against XE T linked to AffiGel 10 (Affi-Ab) adsorbed both XET and XET-XG complex but not [H-3]XG alone. XET immobilized onto Affi-Ab was able to bind [ H-3]XG and catalyze transglycosylation in presence of XGOs.