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- W2318086784 abstract "Nitronate monooxygenase is a flavin-dependent enzyme that catalyzes the denitrification of propionate 3-nitronate (P3N) and other alkyl nitronates. The enzyme was previously known as 2-nitropropane dioxygenase, until its reclassification in 2010 by the IUBMB. Physiologically, the monooxygenase from fungi protects the organism from the environmental occurrence of P3N, which shuts down the Krebs cycle by inactivating succinate dehydrogenase and fumarase. The inhibition of these enzymes yields severe neurological disorders or death. Here, we have used for the first time steady-state and rapid kinetics, viscosity and pH effects, and time-resolved absorbance spectroscopy of the enzyme in turnover with P3N and the substrate analogue ethyl nitronate (EN) to elucidate the mechanism of the reaction. A transient increase in absorbance at ∼300 nm, never reported before, was seen during steady-state turnover of the enzyme with P3N and oxygen, with no concomitant changes between 400 and 600 nm. The transient species was not detected when oxygen was absent. Anaerobic reduction of the enzyme with P3N yielded anionic flavosemiquinone and was fast (e.g., ≥1900 s(-1)). Steady-state kinetics demonstrated that oxygen reacts before the release of the product of P3N oxidation from the enzyme. No pH effects were seen with P3N on kcat/Km, kcat/Koxygen, and kcat; in contrast, with EN, the kcat/Km and kcat decreased with increasing pH defining two plateaus and a pKa ∼ 8.0. Solvent viscosity at the pH optima suggested product release as being partially controlling the overall rate of turnover with the physiological substrate and its analogue. A mechanism that satisfies the kinetic results is proposed." @default.
- W2318086784 created "2016-06-24" @default.
- W2318086784 creator A5022045497 @default.
- W2318086784 creator A5032766595 @default.
- W2318086784 date "2013-04-05" @default.
- W2318086784 modified "2023-10-14" @default.
- W2318086784 title "Evidence for a Transient Peroxynitro Acid in the Reaction Catalyzed by Nitronate Monooxygenase with Propionate 3-Nitronate" @default.
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- W2318086784 doi "https://doi.org/10.1021/bi400030d" @default.
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