Mechanism-based inhibitors of dopamine beta-hydroxylase: inhibition by 2-bromo-3-(p-hydroxyphenyl)-1-propene.
The compound 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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001051:Apoenzymes / Q000037:antagonists & inhibitors; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology; D003300:Copper / Q000494:pharmacology; D004299:Dopamine beta-Hydroxylase / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010636:Phenols / Q000138:chemical synthesis / Q000494:pharmacology*
C040825:2-bromo-(4-hydroxyphenyl)-1-propene; D001051:Apoenzymes; D010636:Phenols; D003300:Copper; D004299:Dopamine beta-Hydroxylase