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 ~3 s-1 at 21 °C. Analysis of 5'-deoxyadenosine isolated from a reaction mixture prepared in 2H2O did not contain deuterium-a 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- 2H4]HEH, had acquired at least one 2H 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.
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