Peptidylglycine ?-amidating monooxygenase (PAM) and dopamine ?-monooxygenase (DBM) are members of a small group of copper containing monooxygenases that carry out important neurological and endocrinological functions. PAM catalyzes the C-terminal amidation of neuropeptides to their biologically active, mature forms. This critical post translational modification imparts functionality to the neuropeptides in the regulation of a wide variety of processes, including embryonic development, stress response, pain, digestion, bone mineralization, sexual response, blood volume and pressure, and many others. Elevated expression of PAM is detectable in many endocrine tumors, particularly prostate cancers, where amidated peptides are believed to act as mediators of tumor growth. DBM and its homologue tyramine monooxygenase (TM) catalyze the hydroxylation of dopamine and tyramine to nor-adrenalin and octopamine respectively, and therefore fulfill pivotal roles in neurotransmitter biosynthesis. These enzymes are important for two major reasons. First they catalyze essential neurophysiological reactions which in the case of PAM are of pharmacological interest, and second, they react via novel chemical mechanisms which are incompletely understood. We propose a program of study aimed at further investigating the reaction mechanisms, and the underlying chemistry on which they are based. We plan to study (i) the nature of the copper-dioxygen intermediates using advanced spectroscopic approaches, (ii) the role of essential catalytic residues, and in particular the enigmatic copper-ligand M314, (iii) the mechanism of electron transfer between the two mononuclear copper centers, and the ET pathways by which these sites are connected, and (iv) the structure of the recently discovered heterotrimetallic Fe, Zn, Ca center at the active center of the PAL domain of the bifunctional PAM protein. These studies will place the catalytic chemistry on a firm mechanistic basis, and advance the goal of targeting the enzymes, their substrates and products as pharamacological agents.
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