This research proposal deals with mechanistic studies of hydroxylases, in particular, dopamine-beta-hydroxylase (DBetaH) from adrenal medulla.
Our aim i s to understand the details of the mechanism so that we can develop compounds to control in vivo the levels of noradrenaline, a nerve transmitter, the product of the enzymatic oxidation of dopamine catalyzed by DBetaH. Studying the mechanism of the hydroxylation reaction will be approached in several ways: 1) Define the role of Cu in the reaction cycle especially with regard to the stoichiometry of Cu at the active site during catalysis and the flow of electrons from reductant to metal ion to oxygen. EPR and NMR techniques will be employed. 2) This laboratory was the first to describe a suicide substrate for DBetaH. These studies will be continued and pursued with other compounds that may also be mechanism-based, active-site directed inhibitors of this enzyme. 3) The reactivity of DBetaH will be explored in vivo in isolated chromaffin granules. We have shown that the enzymatic activity of both soluble and membrane-bound enzyme can be followed by monitoring conversion of 13C-tyramine to 13C-octopamine by 13C-NMR. Suicide inhibition of the enzyme will be monitored in vivo by this technique as well as the role of reductants for the enzymatic reaction in the intact chromaffin granules. Several neurological disorders are characterized by an elevated level of noradrenaline. Over production of noradrenaline is associated with the human adrenal phaeochromocytoma tumor. Specific inhibition of an enzyme using the technique of suicide substrate inactivation is one important approach in the search for specific drugs to alter noradrenaline production in vivo.
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