Abstract
Dopamine β-hydroxylase is inactivated by phenyl-, phenethyl-, benzyl-, and methylhydrazine, but not by hydrazine itslef. With phenyl-, methyl-, and phenethylhydrazine, the rate of inactivation decreases in the presence of ascorbate and increases in the presence of tyramine. Reduction of the enzyme-bound copper occurs with all of the hydrazines tested. In the presence of the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone the carbon-centered radicals generated from each compound are trapped. This is consistent with reduction of the enzyme-bound copper by the hydrazine-containing compounds, resulting in formation of the hydrazine cation radical. Homolytic cleavage of the carbon-nitrogen bond then generates a carbon-centered radical which reacts with the enzyme, resulting in inactivation. Inactivation with [14C]phenylhydrazine results in the incorporation of 0.94 molecule of label per enzyme subunit. Benzylhydrazine behaves as a mechanism-based inhibitor of the enzyme. Both benzyl- and phenethylhydrazine are substrates for dopamine β-hydroxylase. The second-order rate constant for inactivation of dopamine β-hydroxylase by benzylhydrazine in the presence of ascorbate is increased about 4-fold when the benzylic hydrogens are replaced with deuterium. The apparent V(max) shows an observed deuterium kinetic isotope effect of 13 ± 2. The partition ratio for product formation versus inactivation is 11-fold less for α,α-d2-benzylhydrazine. These results are interpreted in terms of a model where inactivation is due to abstraction of an electron from nitrogen instead of abstraction of a hydrogen atom from the benzylic carbon.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 4510-4518 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 261 |
| Issue number | 10 |
| State | Published - 1986 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
The mechanism of inactivation of dopamine β-hydroxylase by hydrazines. / Fitzpatrick, Paul F; Villafranca, J. J.
In: Journal of Biological Chemistry, Vol. 261, No. 10, 1986, p. 4510-4518.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The mechanism of inactivation of dopamine β-hydroxylase by hydrazines
AU - Fitzpatrick, Paul F
AU - Villafranca, J. J.
PY - 1986
Y1 - 1986
N2 - Dopamine β-hydroxylase is inactivated by phenyl-, phenethyl-, benzyl-, and methylhydrazine, but not by hydrazine itslef. With phenyl-, methyl-, and phenethylhydrazine, the rate of inactivation decreases in the presence of ascorbate and increases in the presence of tyramine. Reduction of the enzyme-bound copper occurs with all of the hydrazines tested. In the presence of the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone the carbon-centered radicals generated from each compound are trapped. This is consistent with reduction of the enzyme-bound copper by the hydrazine-containing compounds, resulting in formation of the hydrazine cation radical. Homolytic cleavage of the carbon-nitrogen bond then generates a carbon-centered radical which reacts with the enzyme, resulting in inactivation. Inactivation with [14C]phenylhydrazine results in the incorporation of 0.94 molecule of label per enzyme subunit. Benzylhydrazine behaves as a mechanism-based inhibitor of the enzyme. Both benzyl- and phenethylhydrazine are substrates for dopamine β-hydroxylase. The second-order rate constant for inactivation of dopamine β-hydroxylase by benzylhydrazine in the presence of ascorbate is increased about 4-fold when the benzylic hydrogens are replaced with deuterium. The apparent V(max) shows an observed deuterium kinetic isotope effect of 13 ± 2. The partition ratio for product formation versus inactivation is 11-fold less for α,α-d2-benzylhydrazine. These results are interpreted in terms of a model where inactivation is due to abstraction of an electron from nitrogen instead of abstraction of a hydrogen atom from the benzylic carbon.
AB - Dopamine β-hydroxylase is inactivated by phenyl-, phenethyl-, benzyl-, and methylhydrazine, but not by hydrazine itslef. With phenyl-, methyl-, and phenethylhydrazine, the rate of inactivation decreases in the presence of ascorbate and increases in the presence of tyramine. Reduction of the enzyme-bound copper occurs with all of the hydrazines tested. In the presence of the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone the carbon-centered radicals generated from each compound are trapped. This is consistent with reduction of the enzyme-bound copper by the hydrazine-containing compounds, resulting in formation of the hydrazine cation radical. Homolytic cleavage of the carbon-nitrogen bond then generates a carbon-centered radical which reacts with the enzyme, resulting in inactivation. Inactivation with [14C]phenylhydrazine results in the incorporation of 0.94 molecule of label per enzyme subunit. Benzylhydrazine behaves as a mechanism-based inhibitor of the enzyme. Both benzyl- and phenethylhydrazine are substrates for dopamine β-hydroxylase. The second-order rate constant for inactivation of dopamine β-hydroxylase by benzylhydrazine in the presence of ascorbate is increased about 4-fold when the benzylic hydrogens are replaced with deuterium. The apparent V(max) shows an observed deuterium kinetic isotope effect of 13 ± 2. The partition ratio for product formation versus inactivation is 11-fold less for α,α-d2-benzylhydrazine. These results are interpreted in terms of a model where inactivation is due to abstraction of an electron from nitrogen instead of abstraction of a hydrogen atom from the benzylic carbon.
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UR - http://www.scopus.com/inward/citedby.url?scp=0023002228&partnerID=8YFLogxK
M3 - Article
C2 - 3007460
AN - SCOPUS:0023002228
VL - 261
SP - 4510
EP - 4518
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 10
ER -