Mechanisms of Flavoprotein Oxidases

摘要

Mechanistic studies have been carried out on two flavoprotein oxidases, D-amino acid oxidase and nitroalkane oxidase. D-Amino acid oxidase is one of several amino acid oxidases proposed to initiate substrate oxidation via formation of a carbanion. Intrinsic kinetic isotope effects were determined with the slow substrate D-serine: ~DV/K = 4.5 +- 0.2, ?TV/K = 8.6 +- 0.1, and ~(D2O)V/K = 0.9 +- 0.12. The ~(15)V/K value decreases with increased pH and increases in D_2O, establishing that the amino group of the amino acid substrate must be unprotonated for catalysis. Correcting for the protonation state of the substrate yields a pH and solvent independent value of 0.997 +- 0.001, a value consistent with a hydride transfer mechanism rather than a carbanion. In contrast, results obtained with nitroalkane oxidase are consistent with carbon-hydrogen bond cleavage via a carbanion. As isolated, the FAD in this enzyme is in the form of an inactive 5-nitrobutyl-FAD. This can be converted to the active FAD-containing enzyme. The FAD can be converted to the adduct by incubation with a mixture of neutral and anionic nitroethane. Results of pH and kinetic isotope effects are consistent with the neutral form of the nitroalkane being the substrate.