Functionalization of the beta-
carbon of
phenethylamines has been shown to produce a new class of substrate/inhibitor of
dopamine beta-monooxygenase. Whereas both beta-hydroxy- and
beta- chlorophenethylamine are converted to
alpha-aminoacetophenone at comparable rates, only the latter conversion is accompanied by concomitant
enzyme inactivation ( Klinman , J. P., and Krueger , M. (1982) Biochemistry 21, 67-75). In the present study, the nature of the reactive intermediates leading to
dopamine beta-monooxygenase inactivation by
beta- chlorophenethylamine has been investigated employing kinetic
deuterium isotope effects and
oxygen- 18 labeling as tools. Mechanistically significant findings presented herein include: 1) an analysis of primary
deuterium isotope effects on turnover, indicating major differences in rate-determining steps for beta-chloro- and
beta- hydroxyphenethylamine hydroxylation, Dkcat = 6.1 and 1.0, respectively; 2) evidence that
dehydration of the gem-diol derived from
oxygen- 18-labeled
beta- hydroxyphenethylamine hydroxylation occurs in a random manner, attributed to dissociation of
enzyme-bound gem-diol prior to
alpha-aminoacetophenone formation; 3) the observation of a
deuterium isotope effect for
beta- chlorophenethylamine inactivation, Dkinact = 3.7, implicating C--H bond cleavage in the inactivation process; and 4) the demonstration that
alpha-aminoacetophenone can replace
ascorbic acid as exogenous
reductant in the hydroxylation of
tyramine. As discussed, these findings support the intermediacy of
enzyme-bound
alpha-aminoacetophenone in
beta- chlorophenethylamine inactivation, and lead us to propose an intramolecular redox reaction to generate a
ketone-derived radical
cation as the
dopamine beta-monooxygenase-inactivating species.