The Mechanism of Action of Ethanolamine Ammonia-Lyase, a B12-dependent Enzyme

The previous observation (Babior, B. M., J. Biol. Chem., 244, 449 (1969)) that the tritium isotope effect for the coenzyme B12-dependent deamination of ethanolamine was smaller than the deuterium isotope effect prompted a reexamination of the various isotope effects observed in this reaction. Measurements of the rates of deamination of [1-D]- and [1,1-D2]ethanolamine showed that no secondary isotope effect was detectable within the limits of experimental error. Activation parameters for the deamination of unlabeled ethanolamine and [1,1-D2]ethanolamine at 23° were as follows: for the unlabeled substrate, Ea = 3.9 kcal per mole and S = -9.7 e.u.; for the deuterated substrate, Ea = 5.5 kcal per mole and S = -9.4 e.u. The similarity of these values implies that the rate-limiting step does not change on passing from the unlabeled to the deuterium-labeled substrate. Measurement of the tritium isotope effect for each of the two individual hydrogen transfer steps of the reaction gave the following results: for the first step (transfer of hydrogen from substrate to coenzyme), kt/kh = 4.7; for the second step (transfer of hydrogen from coenzyme to product), kt/kh = 160 and kt/kd = 22. The over-all deuterium isotope effect was found to be 7.4, confirming previous results. From these and other observations the following conclusions were drawn: (a) the rate-limiting step is the transfer of hydrogen from coenzyme to product; (b) an irreversible step occurs between the first hydrogen transfer step and the second; and (c) exchange between free and enzyme-bound coenzyme B12 during the course of the reaction is slow.