The mechanism of inactivation of dopamine β-hydroxylase by hydrazines

Paul F Fitzpatrick, J. J. Villafranca

Research output: Contribution to journalArticle

30 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)4510-4518
Number of pages9
JournalJournal of Biological Chemistry
Volume261
Issue number10
StatePublished - 1986
Externally publishedYes

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hydrazine
Hydrazines
Mixed Function Oxygenases
Dopamine
Carbon
Phenelzine
Deuterium
Enzymes
Copper
Hydrogen
Monomethylhydrazine
Nitrogen
Tyramine
Enzyme Inhibitors
Isotopes
Oxides
Cations
Labels
Rate constants
Electrons

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 journalArticle

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