pH and deuterium isotope effects on the reaction of trimethylamine dehydrogenase with dimethylamine.

The flavoprotein trimethylamine dehydrogenase is a member of a small class of flavoproteins that catalyze amine oxidation and transfer the electrons through an Fe/S center to an external oxidant. The mechanism of amine oxidation by this family of enzymes has not been established. Here, we describe the use of pH and kinetic isotope effects with the slow substrate dimethylamine to study the mechanism. The data are consistent with the neutral amine being the form of the substrate that binds productively at the pH optimum, since the pKa seen in the kcat/Kamine pH profile for a group that must be unprotonated matches the pKa of dimethylamine. The D(kcat/Kamine) value decreases to unity as the pH decreases. This suggests the presence of an alternative pathway at low pH, in which the protonated substrate binds and is then deprotonated by an active-site residue prior to oxidation. The kcat and Dkcat values both decrease to limiting values at low pH with similar pKa values. This is consistent with a step other than amine oxidation becoming rate-limiting for turnover.