Mechanism-based inhibitors of dopamine .beta.-hydroxylase: inhibition by 2-bromo-3-(p-hydroxyphenyl)-1-propene

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Abstract
2-Bromo-3-(p-hydroxyphenyl)-1-propene (I) is a substrate of dopamine .beta.-hydroxylase from adrenal medulla, and the product has been identified by mass spectrometry as 2-bromo-3-hydroxy-3-(p-hydroxyphenyl)-1-propene (II). Compound I also inactivates dopamine .beta.-hydroxylase in a mechanism-based fashion. Thus, in acetate buffer at pH 5.0, inactivation by I exhibits saturation kinetics with a KD = 4.5 .mu.M and kinact = 0.09 min-1. The inactivation is strictly dependent on O2 and a reducing agent (ascorbate or ferrocyanide) and is irreversible with no reactivation occurring upon prolonged dialysis or passage through a gel filtration column. The observed rate of inactivation at [I]= 4.5 mM (pH 5.5) increases from 0.045 to 0.17 min-1 when [O2] is increased from 0.25 to 1.2 mM. Norepinephrine affords competitive protection against inactivation of enzyme by I. In initial velocity experiments, I is a linear competitive inhibitor vs. tyramine. The log Kis vs. pH profile is flat while the log kinact vs. pH profile has an inflection corresponding to a group with a pKa of 5.7 .+-. 0.1. These data demonstrate that an enzymic group in its protonated form is involved in the inactivation reaction and not in the binding of I to the enzyme. In addition, inactivation requires a catalytically competent enzyme, inasmuch as no inactivation occurs when Cu2+-free enzyme is incubated with I in the presence of ascorbate and O2. At pH 5.5, as Cu2+ is added to apoenzyme or to enzyme of low Cu content, the rate at inactivation at saturating I increases and reaches a maximal value at 8 Cu2+ per tetramer. Because inactivation is strictly dependent on catalysis and follows saturation kinetics, i.e., a Michaelis complex is formed, 8 Cu per tetramer are required for fully active enzyme and 4 active sites each containing 2 Cu2+ ions exist per tetramer. On the basis of these data, a mechanism of inactivation is suggested that involves enzyme-catalyzed activation of I to a reactive intermediate or to II followed by formation of a covalent adduct with the enzyme.