Hydrogen Atom Exchange between 5‘-Deoxyadenosine and Hydroxyethylhydrazine during the Single Turnover Inactivation of Ethanolamine Ammonia-Lyase

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.

Isotope Effects in the Transient Phases of the Reaction Catalyzed by Ethanolamine Ammonia-Lyase: Determination of the Number of Exchangeable Hydrogens in the Enzyme−Cofactor Complex

“…The most straightforward interpretation of the results regarding the onset of the 2 H KIE on reformation of coenzyme B 12 is that the only group being loaded with deuterium in experiments with unlabeled cofactor and deuterated substrates is the 5′methylene of the adenosyl moiety of the cofactor. This interpretation is consistent with results from studies of EAL with the single turnover inactivator, hydroxyethylhydrazine, which show that there is direct hydrogen exchange between the inactivator and 5′-deoxyadenosine without an intermediate carrier (40). Mass spectrometry was used previously to determine that the total pool (including the substrate) of exchangeable hydrogens in methylmalonyl CoA mutase was three (41).…”

Hydrazine Cation Radical in the Active Site of Ethanolamine Ammonia-Lyase: Mechanism-Based Inactivation by Hydroxyethylhydrazine

“…The presence of equimolar quantities of acetaldehyde and 5′-deoxyadenosine rules out multiple turnovers of HEH. Studies with [1,1,2,2-2 H 4 ]HEH, however, indicate that the first step is reversible (42). The HEH radical can eliminate the hydrazine cation radical leaving the enol of acetaldehyde (Scheme 2, path A) or rearrange to a product radical before fragmentation (Scheme 2, path B).…”

Chemistry and Enzymology of Vitamin B12

“…312,313 Hydroxyethylhydrazine (HEH), an analogue of 2-aminoethanol, has been found to be a mechanismbased inhibitor of EAL. 317,318 Incubation of EAL with excess AdoCbl and HEH leads to rapid loss (k ) 2.7 ( 0.3 s -1 ) of activity and the formation of about 6 equiv each of cob(II)alamin, 5′-deoxyadenosine, and acetaldehyde. Although earlier work had suggested that the R 6 β 6 EAL oligomer had two active sites, the stoichiometry of the reaction of EAL with HEH and AdoCbl indicates six active sites per molecule.…”