Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Deuterium, solvent, and (15)N 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 b(2) 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.