Copper-containing amine oxidases are widely distributed in nature and are involved in the metabolism of biogenic primary amines, which have a variety of functions in the cardiovascular, gastrointestinal, and nervous systems; more generally, primary amines are implicated in the control of cell proliferation and differentiation. Amine oxidases are also responsible for the crosslinking of connective tissue structural proteins (elastin and collagen) in aorta, lung, and cartilage. The principal objectives of the proposed research are: (1) to elucidate the molecular structure(s) of copper-containing amine oxidases, particularly of the active sites; (2) to determine the role of copper, the organic cofactor, and any copper-cofactor interactions in catalytic mechanisms of amine oxidases; (3) to develop detailed structure/reactivity/function correlations among amine oxidases. Several experimental approaches will be employed. A concerted effort is planned to obtain the X-ray crystal structure of at least one amine oxidase. Concurrently, peptides derived from the active sites of several enzymes and containing derivatives of the cofactor will be prepared and structurally characterized. Resonance Raman, EPR, pulsed EPR, and ENODR spectroscopy will be used to probe the structure of the organic cofactor in various oxidation states and its possible interactions with the copper ions. It is also proposed to exploit the properties of Cu(II) as a built-in probe of the active site by using several different spectroscopic techniques, including resonance Raman, EPR (CW and pulsed), optical (absorption, circular dichroism, magnetic circular dichroism) spectroscopy and solvent nuclear magnetic relaxation dispersion measurements. We intend to intensively study the resting, reduced, copper-depleted, and metal-substituted forms of several amine oxidases. Stopped-flow and low-temperature kinetics studies will be carried out in order to further define mechanistic role(s) for copper. Experiments designed to trap or stabilize catalytic intermediates are proposed in order to structurally characterize any such intermediates. Substrate-reduced forms of the enzymes will receive particular attention. Spectroscopic and kinetics studies of the interactions of amine oxidases with small molecules (inhibitors, substrate analogs, products) are planned. Collectively, the results of these experiments should provide significant new information concerning the structure and function of this diverse group of enzymes.
Showing the most recent 10 out of 64 publications
