DOI:
Authors:
Vahe Bandarian, Russell R. Poyner, George H. Reed
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 approximately 3 s(-)(1) at 21 degrees C. Analysis of 5′-deoxyadenosine isolated from a reaction mixture prepared in (2)H(2)O 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-(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.