Colombo G, Rajashekhar B, Giedroc D P, Villafranca J J
Biochemistry. 1984 Jul 31;23(16):3590-8. doi: 10.1021/bi00311a004.
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)
