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. Amine oxidases are also responsible for the crosslinking of connective tissue structural proteins (elastin and collagen). Recently, amine oxidases have emerged as the first examples of what may prove to be a wholly new class enzyme, that is, where a post-translationally modified amino acid side chain is present in the active site and has a redox role in catalysis. The principal goals are to elucidate the molecular structures, especially of the active sites, and the catalytic mechanisms of copper-containing amine oxidases. Coordinated structural, spectroscopic, and mechanistic experiments will be carried out concurrently. Specific goals are to: (1) complete the crystal structure of oxidized pea seedling amine oxidase and prepare crystalline samples of the reduced Cu(I) form and complexes with substrates, or substrate analogues, and inhibitors, for X-ray diffraction measurements; (2) define in detail the mechanism of amine oxidases, including the role(s) of copper, and rigorously test the hypothesis that the Cu(I)- semiquinone state is the catalytic intermediate that reacts with oxygen; (3) elucidate the electronic structures and related structural properties of the Cu(II) sites in both the oxidized and substrate-reduced states, and in the Cu(I)- semiquinone state. To complement the X-ray analysis, it is planned to isolate and sequence active-site peptides. Magnetic circular dichroism, X-ray absorption (EXAFS), resonance Raman, ENDOR, and cw- and pulsed-EPR spectroscopy will be used to characterize the copper sites, including the Cu(I)-semiquinone state. The formation and subsequent reactions of the Cu(I)-semiquinone state will be investigated by both temperature-jump and stopped-flow circular dichroism. Additional mechanistic information will come from spectroscopic and kinetics experiments with """"""""slow"""""""" amine substrates and the reactions with NO and H2O2. Parallel spectroscopic experiments on methylamine dehydrogenase and galactose oxidase are planned. Comparative studies among amine oxidases, methylamine dehydrogenases, and galactose oxidase will provide additional insights into structure- function relationships among enzymes containing post-translationally modified, redox-active amino acids in their active sites.
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