Abstract
Catalytic cleavage reactions by glucoamylase or phosphorylase b were monitored directly on an amylopectin-immobilized 27MHz quartz-crystal microbalance (QCM). As a first example, we could follow kinetically the enzyme binding to the substrate binding (kon) and dissociation rate constants (koff) and intramolecular hydrolysis rate constant (kcat) of glucan hydrolysis by glucoamylase by detecting directly the formation and decomposition of the enzyme-substrate (ES) complex as mass changes. Secondly, glucan phosphorolysis by phosphorylase b was directly observed by two methods through reactions on a QCM. All kinetic parameters for the enzyme binding to the substrate kon and koff, and dissociation constant, Kd, the AMP binding to the enzyme as activator (KAMP), and the catalytic rate constant (kcat) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics in the bulk solution.
