The reaction of trimethylamine dehydrogenase with diethylmethylamine.

The reductive half-reaction of trimethylamine dehydrogenase has been studied using the substrate diethylmethylamine over the pH range 6-10. It is found that the reaction occurs with three distinct and, under most conditions, fully resolved kinetic phases. The hyperbolic substrate concentration dependence of the observed rate constant for the fast kinetic phase is consistent with a two-step scheme in which free enzyme and substrate are in rapid equilibrium with an enzyme-substrate Michaelis complex, which then reacts to produce reduced flavin. The pH dependence of the limiting rate constant for the fast phase of the reaction (klim) exhibits a pKa value of 7.9, whereas klim/Kd exhibits a pKa value of 8.7. The rate constant for the intermediate kinetic phase, which reflects intramolecular electron transfer, is controlled by an ionizable group exhibiting a pKa value of 7.3. The equilibrium distribution of reducing equivalents between the flavin and iron-sulfur center of the enzyme at the end of the intermediate phase is controlled by a different ionizable group exhibiting a pKa value of approximately 6. An overall reductive half-reaction kinetic mechanism is proposed involving formation and decay of a covalent substrate-flavin intermediate, with intrinsically rapid intramolecular electron transfer limited by the rate of decay of the covalent adduct. Under conditions of excess substrate, product release is followed by binding of a second substrate molecule, which results in full development of the spectral properties diagnostic of the spin-interacting state.