Dopamine beta-monooxygenase (DbetaM) 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). Because of the vital physiologic roles played by norepinephrine, DbetaM is a logical target in establishing a molecular basis for our understanding of neurologic disorders and in developing drug therapies. The goal of this proposal is to elucidate the molecular mechanisms of catalysis and regulation for DbetaM from bovine adrenal medulla. A long term objective is to integrate the properties of the isolated enzyme into a detailed understanding of its structure and function within adrenal chromaffin granules.
Specific aims are in three categories. I. Reaction Mechanism: (a) The nature of reactive intermediates in enzyme turnover and inhibition will be pursued to include: (i) detection of a Cu(11)-OOH species, postulated to form prior to substrate hydroxylation; (ii) demonstration of the reduction of enzymic cupric ion at the level of the E-P complex, rather than free enzyme; (iii) elaboration of the oxygen intermediate leading to hydrogen abstraction from substrate; and (iv) distinction between species leading to enzyme turnover vs inactivation with cresols as mechanism based inhibitors. (b) Differentiation of the properties for copper binding of each of the two metal sites per subunit will be attempted. II. Structure and Regulation: Experiments will proceed in several directions, which include: (a) expression and mutagenesis of our recently cloned full length c-DNA; (b) preparation of and attempted crystallization of carbohydrate free enzyme; (c) identification of the membrane anchor for the membranous form of DbetaM; (d) elaboration of the pathway for the biogenesis of soluble vs membrane bound enzyme; (e) further characterization of a proposed allosteric regulation of enzyme by ascorbate; and (f) affinity labelling of the fumarate activator site. III. Relationship of DbetaM Properties In Vitro to its Function in Chromaffin Granules: Evidence has been presented for dual pathways in the reduction of membrane bound vs soluble enzyme within the intravesicular space of chromaffin granules. The detailed role of internal and external reductants in the turnover of DbetaM in chromaffin granule ghosts will be pursued.
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