QUINOENZYMES--BIOGENESIS STRUCTURE AND FUNCTION

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During the past five years a new class of proteins has been described, designated quinoproteins. These proteins, which contain their cofactor within their polypeptide backbone, reveal a novel strategy for the generation of redox cofactors in biology. Utilizing a combination of chemical, biochemical and molecular biological approaches, multifaceted studies of these systems will be pursued. A detailed investigation of the biogenesis of the cofactor topa quinone (TPQ) in the copper amine oxidases (CAO's) is planned, with the goal of understanding how the CAO's are capable of catalyzing "self processing". Data available thus far implicate the active site copper of CAO's in both cofactor biogenesis and catalytic turnover. Although formally a member of the CAO family of proteins, lysyl oxidase (LO) differs by virtue of its reduced size and lack of significant sequence homology. Preliminary data from this laboratory indicate that while LO contains a quino-structure, this is different from structures seen previously in other proteins. A large number of experiments are planned, involving characterization of active site derived peptides and the synthesis and comparative study of model compounds. Sequence homology among four cloned and sequenced eukaryotic CAO's indicates a conserved structural motif toward the C-terminus of protein; this structural motif contains both the TPQ consensus sequence and the putative histidine ligands to copper. The gene for yeast amine oxidase from Hansenula polymorpha (YAO) will be modified for expression of the C-terminal domain, with the goal of determining whether cofactor biogenesis can occur within this domain. Efforts currently underway, to obtain an X-ray structure for YAO will be continued and eventually extended to mutant forms of YAO. Solution studies, focused on both an amine oxidase from bovine plasma and YAO, will address a number of questions which include the role of the active site consensus sequence in determining substrate specificity, the nature of the active site base catalyzing substrate oxidation, and the chemical mechanism of the half reaction involving reduction of dioxygen to hydrogen peroxide. Given their wide ranging physiologic functions which include the oxidative removal of biogenic amines from the blood stream and the cross-linking of collagen and elastin, the quinoproteins under study are directly related to human health.