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 ~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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 12403-12407 |
| Number of pages | 5 |
| Journal | Biochemistry |
| Volume | 38 |
| Issue number | 38 |
| DOIs | |
| State | Published - Sep 21 1999 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Hydrogen atom exchange between 5'-deoxyadenosine and hydroxyethylhydrazine during the single turnover inactivation of ethanolamine ammonia-lyase. / Bandarian, Vahe; Poyner, Russell R.; Reed, George H.
In: Biochemistry, Vol. 38, No. 38, 21.09.1999, p. 12403-12407.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Hydrogen atom exchange between 5'-deoxyadenosine and hydroxyethylhydrazine during the single turnover inactivation of ethanolamine ammonia-lyase
AU - Bandarian, Vahe
AU - Poyner, Russell R.
AU - Reed, George H.
PY - 1999/9/21
Y1 - 1999/9/21
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0033592313&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033592313&partnerID=8YFLogxK
U2 - 10.1021/bi9906219
DO - 10.1021/bi9906219
M3 - Article
C2 - 10493808
AN - SCOPUS:0033592313
VL - 38
SP - 12403
EP - 12407
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 38
ER -