The mechanism of inactivation of dopamine beta-hydroxylase by hydrazines.
Dopamine beta-hydroxylase is inactivated by phenyl-, phenethyl-, benzyl-, and methylhydrazine, but not by hydrazine itself. 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 alpha-(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 beta-hydroxylase. The second-order rate constant for inactivation of dopamine beta-hydroxylase by benzylhydrazine in the presence of ascorbate is increased about 4-fold when the benzylic hydrogens are replaced with deuterium. The apparent Vmax shows an observed deuterium kinetic isotope effect of 13 +/- 2. The partition ratio for product formation versus inactivation is 11-fold less for alpha,alpha-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.
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004299:Dopamine beta-Hydroxylase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D006834:Hydrazines / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013329:Structure-Activity Relationship
D006834:Hydrazines; C029424:hydrazine; D004299:Dopamine beta-Hydroxylase