This work focuses on three copper-containing monooxygenases that play an important role in the biosynthesis of neuroregulatory hormones. Dopamine- beta-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenalin in the sympathetic nervous system and is associated with affective disorders, clinical depression and neuroblastoma. Phenylalanine hydroxylase (PAH) synthesizes tyrosine from phenylalanine, and genetic deficiencies in PAH are responsible for phenylketonuria. Peptidly-alpha- amidase (PAM) catalyzes the conversion of glycyl-extended peptides to their active amidated forms, and is responsible for the biosynthesis of essential neuropeptide hormones such as gonadotropin, vasopressin, and oxytocin. These three enzymes belong to the class of copper proteins termed """"""""non- blue"""""""", in which the catalysis is initiated by binding of O2 and substrate at mononuclear Cu(I) centers. The program focuses on understanding the coordination chemistry of the active site metal ions and in particular, the structure and reactivity of the Cu(I) centers, using x-ray absorption and ligand-directed spectroscopic techniques developed in the applicant's laboratory over the last few years. These studies will address important questions such as: (i) What are the structures of the individual Cu atoms? (ii) How is oxygen activated at mononuclear Cu(I) centers? (iii) What is the connectivity between the copper centers and how are electrons shuttled between the sites? Routes to half-met and half-apo DBH will be developed and used to characterize the structure and catalytic role of each copper. Selective binding of CO and azide will be used to probe the coordination at the inequivalent Cu sites, and the chemistry of new spectroscopic reporter ligands such as NO, alkylisocyanides and acetylenes will be developed. Cu(II)-peroxo intermediates will be sought via titration of azido derivatives with H2O2 at low temperature using rapid scan spectrophotometry. These methodologies will be extended to probe the copper environment of PAH and PAM, for which less structural information is available. Since the latter two enzymes have been cloned and can be expressed in moderate-to-high yield, mutagenesis experiments will be undertaken to identify active site Cu-binding residues, and their role in catalysis.
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