Oxidation Mode of Pyranose 2-Oxidase Is Controlled by pH

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• 作者：Methinee Prongjit ; Jeerus Sucharitakul ; Bruce A. Palfey ; Pimchai Chaiyen
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：February 26, 2013
• 年：2013
• 卷：52
• 期：8
• 页码：1437-1445
• 全文大小：490K
• 年卷期：v.52,no.8(February 26, 2013)
• ISSN：1520-4995

文摘

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 卤 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 卤 0.4 s^鈥?.