3-Phenylpropenes as mechanism-based inhibitors of dopamine .beta.-hydroxylase: evidence for a radical mechanism

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Abstract
A series of ring-substituted 3-phenylpropenes was examined as mechanism-based inhibitors for the Cu protein dopamine .beta.-hydroxylase. p-HO-, p-CH3O-, m-HO-, m-CH3O-, p-Br- and p-CN-substituted phenylpropenes all inactivate the enzyme under turnover conditions, requiring ascorbate and O2. Replacement of the benzylic hydrogens in 3-(p-hydroxyphenyl)propene with deuterium results in a kinetic isotope effect of 2.0 on kinact/KO2 but in no effect on the partition ratio, Vmax/kinact, consistent with a stepwise mechanism for hydrogen abstraction and O2 insertion. The partition ratio is unchanged in the pH range from 4.5-7.1. Determination of the kinetics of inactivation and the partition ratios for each of these ring-substituted phenylpropenes has allowed determination of the respective V/KO2 values. A linear free energy plot of these values as a function of .sigma.+ gives a .rho. value of -1.2, while the partition ratios show only a slight decrease upon going from electron-donating to electron-withdrawing groups. The results are consistent with a mechanism for dopamine .beta.-hydroxylase in which a hydrogen atom is abstracted to form a benzylic radical, which then partitions between hydroxylation and enzyme inactivation.