Dopamine Beta-monooxygenase catalyzes the final step in the biosynthesis of norepinephrine from tyrosine. Norepinephrine serves either as a hormone (in the adrenal gland) or a neurotransmitter (in the sympathetic nervous system); it is also a precursor in epinephrine biosynthesis in the adrenal gland. Because of the vital physiologic roles played by norepinephrine, dopamine Beta-monooxygenase is a logical target in establishing a molecular basis for our understanding of neurologic disorders and in developing drug therapies. The goals of this proposal are to determine the molecular mechanisms of dopamine Beta-monooxygenase catalysis and regulation for enzyme from bovine adrenal gland. Steady-state isotope and substituent effects will be investigated to establish the order of addition of reducing equivalents to enzyme, the nature of the E.P complex, and the mode of C-H bond activation. The nature of binding of two coppers per subunit will be investigated, as will the participation of these coppers in substrate/oxygen activation. The catalytic role of copper will be addressed by rapid freeze-quench kinetic studies, to follow the pre-steady state time course for E-Cu(II) reduction by ascorbate and reoxidation by dopamine and oxygen. These studies, together with proposed rapid acid-quench kinetics to monitor the formation of enzyme-bound norepinephrine, should allow us to distinguish among postulated chemical mechanisms. The mechanism of suicide inhibition by Beta-chlorophenethylamine and phenacetaldehyde derivatives will pursued, as probes for the nature of reactive intermediates in enzyme inactivation. Such studies may lead to therapeutic agents, via the specific inactivation of dopamine Beta-monooxygenase. Finally, the properties of dopamine Beta-monooxygenase in chromaffin vesicle ghosts will be studied, with the long-range goal of establishing the extent to which ATPase, the catecholamine carrier, dopamine Beta-monooxygenase, and electron transport proteins influence norepinephrine production in this multi-protein system.
Showing the most recent 10 out of 92 publications
