Kinetic studies of AMP-dependent phosphorolysis of amylopectin catalyzed by phosphorylase b on a 27 MHz quartz-crystal microbalance.
Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.
Nishino
Hidekazu
H
Contribution from the Department of Biomolecular Engineering, Frontier Collaborative Research Center, Tokyo Institute of Technology and CREST, Japan Science and Technology Corporation (JST), 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Murakawa
Akiko
A
Mori
Toshiaki
T
Okahata
Yoshio
Y
D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism*; D000687:Amylopectin / Q000737:chemistry / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002384:Catalysis; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D010763:Phosphorylase b / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation
D000249:Adenosine Monophosphate; D000687:Amylopectin; D010763:Phosphorylase b