The role of copper in topa quinone biogenesis and catalysis, as probed by azide inhibition of a copper amine oxidase from yeast

All known copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanin e quinone (TPQ) as a redox cofactor. TPQ is derived posttranslationally fro m a specific tyrosine residue within the protein itself, and is utilized by the enzyme to oxidize amines to aldehydes. Several oxidative mechanisms fo r both turnover and the biogenesis of the cofactor have been proposed in re cent years, which differ mainly in the nature of the interaction of oxygen with the enzyme. In this study, azide is used to probe the role of copper i n catalysis and biogenesis, especially with respect to potential interactio ns between the metal and oxygen. During turnover, it is found that azide is a noncompetitive inhibitor with respect to O-2, most consistent mechanisti cally with oxygen binding off the metal prior to reaction. During biogenesi s, it is found that azide likely prohibits ligation of the precursor tyrosi ne to the copper, thus preventing the formation of this key intermediate. T his result is consistent with previous proposals, where the copper-tyrosine unit is the species that undergoes reaction with O-2. In addition, it is f ound that oxygen consumption is kinetically uncoupled from TPQ formation; t his leads to an expanded kinetic model for biogenesis, with important impli cations for previous results.