Oxidation mode of pyranose 2-oxidase is controlled by pH.
Pyranose 2-oxidase (P2O) from Trametes multicolor is a flavoenzyme that catalyzes the oxidation of d-glucose and other aldopyranose sugars at the C2 position by using O₂ as an electron acceptor to form the corresponding 2-keto-sugars and H₂O₂. In this study, the effects of pH on the oxidative half-reaction of P2O were investigated using stopped-flow spectrophotometry. The results showed that flavin oxidation occurred via different pathways depending on the pH of the environment. At pH values lower than 8.0, reduced P2O reacts with O₂ to form a C4a-hydroperoxyflavin intermediate, leading to elimination of H₂O₂. At pH 8.0 and higher, the majority of the reduced P2O reacts with O₂ via a pathway that does not allow detection of the C4a-hydroperoxyflavin, and flavin oxidation occurs with decreased rate constants upon the rise in pH. The switching between the two modes of P2O oxidation is controlled by protonation of a group which has a pK(a) of 7.6 ± 0.1. Oxidation reactions of reduced P2O under rapid pH change as performed by stopped-flow mixing were different from the same reactions performed with enzyme pre-equilibrated at the same specified pH values, implying that the protonation of the group which controls the mode of flavin oxidation cannot be rapidly equilibrated with outside solvent. Using a double-mixing stopped-flow experiment, a rate constant for proton dissociation from the reaction site was determined to be 21.0 ± 0.4 s⁻¹.
Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Methinee
M
Prongjit
Jeerus
J
Sucharitakul
Bruce A
BA
Palfey
Pimchai
P
Chaiyen
D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011522:Protons; D013053:Spectrophotometry; D055454:Trametes / Q000201:enzymology*
D011522:Protons; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D010100:Oxygen