Hydrogen atom exchange between 5'-deoxyadenosine and hydroxyethylhydrazine during the single turnover inactivation of ethanolamine ammonia-lyase.

V Bandarian, R R Poyner, G H Reed

Index: Biochemistry 38(38) , 12403-7, (1999)

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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.