Kinetic mechanism of pyranose 2-oxidase from trametes multicolor.
Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoprotein oxidase that catalyzes the oxidation of aldopyranoses by molecular oxygen to yield the corresponding 2-keto-aldoses and hydrogen peroxide. P2O is the first enzyme in the class of flavoprotein oxidases, for which a C4a-hydroperoxy-flavin adenine dinucleotide (FAD) intermediate has been detected during the oxidative half-reaction. In this study, the reduction kinetics of P2O by d-glucose and 2-d-d-glucose at pH 7.0 was investigated using stopped-flow techniques. The results indicate that d-glucose binds to the enzyme with a two-step binding process; the first step is the initial complex formation, while the second step is the isomerization to form an active Michaelis complex (E-Fl(ox):G). Interestingly, the complex (E-Fl(ox):G) showed greater absorbance at 395 nm than the oxidized enzyme, and the isomerization process showed a significant inverse isotope effect, implying that the C2-H bond of d-glucose is more rigid in the E-Fl(ox):G complex than in the free form. A large normal primary isotope effect (k(H)/k(D) = 8.84) was detected in the flavin reduction step. Steady-state kinetics at pH 7.0 shows a series of parallel lines. Kinetics of formation and decay of C-4a-hydroperoxy-FAD is the same in absence and presence of 2-keto-d-glucose, implying that the sugar does not bind to P2O during the oxidative half-reaction. This suggests that the kinetic mechanism of P2O is likely to be the ping-pong-type where the sugar product leaves prior to the oxygen reaction. The movement of the active site loop when oxygen is present is proposed to facilitate the release of the sugar product. Correlation between data from pre-steady-state and steady-state kinetics has shown that the overall turnover of the reaction is limited by the steps of flavin reduction and decay of C4a-hydroperoxy-FAD.
Prongjit
Methinee
M
Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Sucharitakul
Jeerus
J
Wongnate
Thanyaporn
T
Haltrich
Dietmar
D
Chaiyen
Pimchai
P