The reaction of trimethylamine dehydrogenase with trimethylamine

The reductive half-reaction of trimethylamine dehydrogenase with its physio logical substrate trimethylamine has been examined by stopped-flow spectros copy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate ox idation process. As in previous work with the slow substrate diethylmethyla mine, the reaction is found to consist of three well resolved kinetic phase s. The observed rate constant for the fast phase exhibits hyperbolic depend ence on the substrate concentration with an extrapolated limiting rate cons tant (k(lim)) greater than 1000 s(-1) at pH above 8.5, 10 degrees C, The ki netic parameter k(lim)/K-d for the fast phase exhibits a bell-shaped pH dep endence, with two pK(a) values of 9.3 +/- 0.1 and 10.0 +/- 0.1 attributed t o a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for k(lim) gives a single pK(a) value of 7.1 +/- 0.2. The observed rate constants for both the inter mediate and slow phases are found to decrease as the substrate concentratio n is increased. The steady-state kinetic behavior of trimethylamine dehydro genase with trimethylamine has also been examined, and is found to be adequ ately described without invoking a second, inhibitory substrate-binding sit e. The present results demonstrate that: (a) substrate must be protonated i n order to bind to the enzyme; (b) an ionization group on the enzyme is inv olved in substrate binding; (c) an active site general base is involved, bu t not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed wit h enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is n ot required to account for excess substrate inhibition, the kinetic behavio r of trimethylamine dehydrogenase can be readily explained in the context o f the known properties of the enzyme.