The Mechanism of Action of Ethanolamine Ammonia-Lyase, a B12-dependent Enzyme: THE PARTICIPATION OF PARAMAGNETIC SPECIES IN THE CATALYTIC DEAMINATION OF 2-AMINOPROPANOL

Abstract

Abstract Ethanolamine ammonia-lyase catalyzed the adenosylcobalamin-dependent deamination of 2-aminopropanol as well as that of ethanolamine. When the enzyme-coenzyme complex was frozen in liquid nitrogen while it was acting on 2-aminopropanol, a large, well defined EPR spectrum was demonstrated. The spectrum consisted of two components: a broad signal at g = 2.34, and an asymmetrical doublet with g values of 2.04 and 1.99. The over-all signal corresponded to about 1.3 spins per active site, the electron density being approximately equally divided between the species producing the g = 2.34 component and the one represented by the asymmetrical doublet. The configuration of the signal was the same regardless of whether the incubation was conducted aerobically or anaerobically. Both components of the signal were saturated by increasing power, the doublet being the more easily saturated component. EPR spectroscopy at two different microwave frequencies (X band and K band) showed that the doublet represented a single paramagnetic species whose signal was split by an interaction with another paramagnetic species in its vicinity. By its location, configuration, and the characteristics of the superimposed hyperfine structure, the g = 2.34 signal was assigned to the unpaired electron of cob(II)alamin. Experiments with isotopically labeled propanolamine permitted the doublet signal to be assigned to the 2-aminopropanol-1-yl radical. The major splitting of the doublet signal was attributed to a dipole-dipole interaction between the 2-amino-propanol-1-yl radical and the unpaired electron on cob(II)-alamin; assuming this explanation to be correct, the distance between the two spins was calculated to be ∼6 A. Rapid mixing, quick freeze experiments showed that the appearance of the paramagnetic species was a first order process with a rate constant of 7 s-1. Since the turnover number for propanolamine is 1 to 2 s-1, the process by which the paramagnetic species are generated is kinetically competent with respect to over-all catalysis. Once formed, the species disappeared with a half-time of about 1 to 2 min. Their disappearance is probably associated with the irreversible destruction of adenosylcobalamin by a side reaction not directly relevant to the catalytic mechanism. These results have been interpreted in terms of a mechanism whereby the early hydrogen transfer steps in the adenosylcobalamin-dependent rearrangement of propanolamine are as follows: [see PDF for equation]