title: Mechanistic studies of topa quinone biogenesis in phenylethylamine oxidase.
abstract: An alternative purification for apophenylethylamine oxidase from Arthrobacter globiformis has been developed, which avoids the use of possible contaminants that may interfere with the topa quinone (TPQ) self-processing reaction. The binding of Cu(II) and the kinetics of TPQ formation in these enzyme preparations have been reinvestigated. Our results show that Cu(II) is not significantly reduced when added to the apoprotein under anaerobic conditions. The Cu(II) EPR and circular dichroism spectra of the initially formed complex are different from the spectra of the mature Cu(II)/TPQ-containing protein, indicating that the active site structure must be altered during TPQ formation. The kinetics we observe are cleanly first-order in protein [measured subsequent to Cu(II) binding] when dioxygen is present in pseudo-first-order excess (k(obs) = 1.5 min(-1)). We found no rate dependence on copper, so long as one copper per subunit was present. This indicates that tyrosine oxidation to give TPQ depends only on the copper that is bound in the active site. These results differ from those originally reported; an alternative mechanism, which involves attack of an activated copper-oxygen species on a tyrosine radical intermediate, is proposed for TPQ formation.
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Ruggiero
authors.forename: J A
authors.initials: JA
authors.lastname: Smith
authors.forename: K
authors.initials: K
authors.lastname: Tanizawa
authors.forename: D M
authors.initials: DM
authors.lastname: Dooley
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000302:isolation & purification* / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology*; D003300:Copper / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics
chemical_list: D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D003300:Copper; C086534:phenylethylamine oxidase; D006631:Amine Oxidase (Copper-Containing)