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

Abstract

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 pK_a seen in the k_cat/K_amine pH profile for a group that must be unprotonated matches the pK_a of dimethylamine. The ^D(k_cat/K_amine) 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 k_cat and ^Dk_cat values both decrease to limiting values at low pH with similar pK_a values. This is consistent with a step other than amine oxidation becoming rate-limiting for turnover.

Keywords: Amine oxidation; Enzyme mechanism; Flavoprotein; Isotope effect; Trimethylamine dehydrogenase; pH effect.