Research Projects
Structures, Mechanisms and Biogenesis of Amine Oxidases
My group is conducting research on the structures, reactivities, regulation, biosynthesis, and functions of several metalloenzymes as well as other areas of bioinorganic chemistry. 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 cross-linking 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 of 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 of our research are to elucidate the molecular structures, the catalytic mechanisms, and the mechanism of post-translation modification of copper-containing amine oxidases. Coordinated structural, spectroscopic, and mechanistic experiments are carried out concurrently. We utilize a wide variety of spectroscopic techniques including magnetic circular dichroism, x-ray absorption (EXAFS), resonance Raman, and cw and pulsed epr. The structure of amine oxidases at atomic resolution are being determined in collaboration with x-ray crystallography groups. The formation and subsequent reactions of intermediates are investigated by both temperature-jump and stopped-flow absorption/circular dichroism. Additional mechanistic information is derived from spectroscopic and kinetics experiments with substrates and reactions with substrate analogues. Comparative studies on amine oxidases, methylamine dehydrogenases, and galactose oxidase provide additional insights into structure-function relationships among enzymes containing post-translationally modified, redox-active amino acids in their active sites. We have recently demonstrated that the two processing events which occur in the maturation of galactose oxidase, the cleavage of a 17 amino acid N-terminal pro-sequence and the formation of the Tyr-Cys cofactor, are self-processing reactions requiring only copper ions and dioxygen.
Personnel:
David Dooley
Keywords:
Bioinorganic, Biochemistry
