A number of medically relevant mammalian proteins have been found to contain novel redox cofactors that are derived from amino acid side chains. These cofactors are 2,4,5 trihydroxyphenylalanyl quinone (TPQ), discovered in the copper amine oxidase family of proteins, and the lysine tyrosyl quinone (LTQ), present in the active site of lysyl oxidases. Two other tyrosine-derived cofactors have also been described in fungi and prokaryotes. These are the protein bound cysteine tyrosyl radical (CTR) in galactose oxidase and the peptide derived pyrroloquinoline quinone (PQQ) produced by a variety of bacteria. A major goal of this proposal is to elucidate the pathway for the biogenesis of each of these tyrosine-derived cofactors and to determine the degree to which common strategies have (or have not) been employed to achieve the very different final cofactor structures. In two instances (TPQ and CTR), evidence has been presented that biogenesis occurs solely by the addition of 02 and Cu 2+ to precursor protein. This is likely to also be true for LTQ, while PQQ production requires a number of gene products. The proposed experimental strategy for studying biogenesis involves the expression of precursor protein or polypeptide (together with the expression of accessory proteins in the case of PQQ), followed by the application of kinetics and spectroscopy to detect and characterize biogenesis intermediates. The impact of cofactor production on cellular physiology may be very significant. In the case of the lysyl oxidases, it is known that many disease states correlate with enzyme activity that will be affected by copper availability and the rate and extent of cofactor production. The cellular roles of the mammalian copper amine oxidases are only now becoming apparent. Experiments are described that address the role of the copper amine oxidase located on the outer surface of the endothelium and the adipocyte. Experimental protocols will focus on the characterization of the endothelial enzyme that we have recently expressed in high yield in insect cells and the use of an adipocyte cell line to follow the impact of copper amine oxidase activity on cellular function.
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