Hydrogen Atom Exchange between 5′-Deoxyadenosine and Hydroxyethylhydrazine during the Single Turnover Inactivation of Ethanolamine Ammonia-Lyase ² Vahe Bandarian, ‡ Russell R. Poyner, and George H. Reed* Institute for Enzyme Research, Graduate School, and Department of Biochemistry, College of Agricultural and Life Sciences, UniVersity of WisconsinsMadison, 1710 UniVersity AVenue, Madison, Wisconsin 53705 ReceiVed March 18, 1999; ReVised Manuscript ReceiVed June 28, 1999 ABSTRACT: The early steps in the single turnover inactivation of ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium by hydroxyethylhydrazine (HEH) have been probed by rapid-mixing sampling techniques, and the destiny of deuterium atoms, present initially in HEH, has been investigated by mass spectrometry. The inactivation reaction produces acetaldehyde, the hydrazine cation radical, 5′- deoxyadenosine, and cob(II)alamin (B 12r ) in amounts stoichiometric with active sites. Rapid-mix freeze- quench EPR spectroscopy and stopped-flow rapid-scan spectrophotometry revealed that the hydrazine cation radical and B 12r appeared at a rate of ∼3s -1 at 21 °C. Analysis of 5′-deoxyadenosine isolated from a reaction mixture prepared in 2 H 2 O did not contain deuteriumsa result which demonstrates that solvent- exchangeable sites are not involved in the hydrogen-transfer processes. In contrast, all of the 5′-deoxyadenosine, isolated from inactivation reactions with [1,1,2,2- 2 H 4 ]HEH, had acquired at least one 2 H from the labeled inactivator. Significant fractions of the 5′-deoxyadenosine acquired two and three deuteriums. These results indicate that hydrogen abstraction from HEH by a radical derived from the cofactor is reversible. The distribution of 5′-deoxyadenosine with one, two, and three deuteriums incorporated and the absence of unlabeled 5′-deoxyadenosine in the product are consistent with a model in which there is direct transfer of hydrogens between the inactivator and the 5′-methyl of 5′- deoxyadenosine. These results reinforce the concept that the 5′-deoxyadenosyl radical is the species that abstracts hydrogen atoms from the substrate in EAL. Ethanolamine ammonia-lyase (EAL; 1 EC 4.3.1.7) catalyzes the coenzyme B 12 -dependent conversion of vicinal amino alcohols to ammonia and the corresponding oxo compound (1-3). An attractive model for the action of enzymes catalyzing coenzyme B 12 -dependent rearrangements is that the coenzyme serves as a radical initiator (4, 5). The 5′- deoxyadenosyl radical, resulting from homolytic cleavage of the cobalt-carbon bond in the cofactor, is thought to initiate the chemical transformations in the substrate directly or indirectly via hydrogen atom abstraction from the substrate molecule. The 5′-deoxyadenosyl radical has, however, eluded direct detection by spectroscopic means. While the presence of substrate-based radicals in reactions catalyzed by EAL is firmly established (6-8), the identity of the immediate progenitor of these radicals is less certain. Experimental observations show that tritium at the 5′-position of the cofactor is discriminated against by a factor of ∼100 in transfer to product (9). This 3 H kinetic isotope effect is much larger than is expected from the deuterium isotope effect in the overall reaction ( D V ≈ 7) (9-12). To rationalize these kinetic isotope effects, the presence of an alternative reaction pathway involving an enzyme-based radical has been proposed (13). This modified scheme removes the need to regenerate the cofactor during every turnover and thus allows “tuning” of the observed 3 H isotope effects to a value more compatible with classical predictions. Some support for the presence of such a protein radical in EAL was found in the form of a solvent exchangeable, or “volatile”, pool of 3 H that is present whenever EAL is allowed to process [1- 3 H]- ethanolamine (14). This pool of 3 H, however, washes out into the product during a chase cycle at the same rate at which 3 H in the cofactor washes out. Although the kinetics of wash out of 3 H from this volatile pool is not compatible with the modified scheme involving the alternative pathway, the identity and number of hydrogen atom abstracting species in EAL is still in question. Incubation of EAL with the substrate analogue, HEH, leads to the complete loss of EAL activity and to the formation of 5′-deoxyadenosine, B 12r , the hydrazine cation radical, and acetaldehyde in amounts that are stoichiometric with respect to the available concentration of active sites (15). The single turnover irreversible inactivation by HEH can be used to probe early steps of EAL catalysis. For example, the fate of deuterium, present initially in the inactivator, can be exam- ined in the absence of multiple turnovers. Thus, the location of deuterium, following the inactivation, identifies the abstracting group. This paper presents results of rapid-mix ² This research was supported by NIH Grant GM35752. * To whom correspondence should be addressed. Phone: (608) 262- 0509. Fax: (608) 265-2904. ‡ Present address: Univ. Michigan, Biophys. Res. Div., Chem. Sci. Bldg., 930 N University, Ann Arbor, MI 48109-1055. 1 Abbreviations: EAL, ethanolamine ammonia-lyase; coenzyme B12, adenosylcobalamin; B12r, cob(II)alamin; EPR, electron paramagnetic resonance; HEH, hydroxyethylhydrazine; Hepes, N-2-hydroxyethylpip- erazine-N′-2-ethanesulfonic acid; GC-MS, gas chromatography-mass spectrometry. 12403 Biochemistry 1999, 38, 12403-12407 10.1021/bi9906219 CCC: $18.00 © 1999 American Chemical Society Published on Web 08/31/1999