Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Deuterium, solvent, and ¹⁵N kinetic isotope effects have been used to probe the mechanisms by which flavoproteins oxidize carbon-oxygen and carbon-nitrogen bonds in amines, hydroxy acids, and alcohols. For the amine oxidases D-amino acid oxidase, N-methyltryptophan oxidase, and tryptophan monooxygenase, D-serine, sarcosine, and alanine are slow substrates for which CH bond cleavage is fully rate limiting. Inverse isotope effects for each of 0.992-0.996 are consistent with a common mechanism involving hydride transfer from the uncharged amine. Computational analyses of possible mechanisms support this conclusion. Deuterium and solvent isotope effects with wild-type and mutant variants of the lactate dehydrogenase flavocytochrome b₂ show that OH and CH bond cleavage are not concerted, but become so in the Y254F enzyme. This is consistent with a highly asynchronous reaction in which OH bond cleavage precedes hydride transfer. The results of Hammett analyses and solvent and deuterium isotope effects support a similar mechanism for alcohol oxidase.