Two related metallomonooxygenases, peptidylglycine alpha- amindating monooxygenase (PAM) and dopamine-beta-hydroxylase (DbetaM), are of particular neurological importance, since they catalyze final steps in the biosynthetic pathways leading to amidated neuropeptides and catecholamine neurotransmitters. This proposal is focused on investigating the catalytic role of copper and zinc, and the relationship between structure and function. Advanced spectroscopic techniques developed in the previous grant period have already led to a model for the active centers of DbetaM. Thee methods will now be applied to the study of wild-type PAM. Comparisons with DbetaM, whose coordination chemistry and spectroscopy are well- developed, will guide the experimental design. The availability of a high-yield expression system in mammalian CHO cell lines has made possible the preparation of a significant number of PAM site-specific mutants, all of which are secreted as viable proteins by the host cell. Kinetic, spectroscopic, and electrochemical studies on these mutants will be undertaken in order to obtain a better understanding of (1) the identity of the metal-binding side chains, (2) the coordination chemistry and catalytic role of the metal centers, and (3) the mechanistic role of non-coordinated active-site residues.
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