2,4,5-Trihydroxyphenylalanine Quinone Biogenesis in the Copper Amine Oxidase from Hansenula polymorpha with the Alternate Metal Nickel

摘要

Copper amine oxidase (CAO) is a dual-functioning enzyme that catalyzes the biosynthesis of a self-derived coenzyme and subsequent oxidative deamination of primary amines.The organic cofactor,2,4,5-trihydroxyphenylalanine quinone (TPQ),is generated from the post-translational modification of an active site tyrosine (Y405) in a reaction shown to be dependent on both molecular oxygen and a mononuclear copper center.Previous investigations of Cu(II)-dependent cofactor formation in the Hansenula polymorpha amine oxidase (HPAO) provided evidence for the coordination of the precursor tyrosine in forming a ligand-to-metal charge transfer complex as a means of activating the tyrosyl ring for direct attack by triplet-state dioxygen.To further delineate the role of the metal in facilitating this complex series of reactions,apo-HPAO was reconstituted with alternate metals of varying reduction potentials and Lewis acidities [Ni(II),Co(II),Mn(II),Fe(II),and Fe(III)] and the consequence of each substitution on TPQ biogenesis examined.Ni(II) was found to support the transformation of the precursor tyrosine to the quinone cofactor to yield a mature enzyme competent for methylamine oxidation.Detailed kinetic analysis of the mechanism of TPQ biogenesis for the Ni(II)-substituted enzyme has led to the proposal of a direct electron transfer from the metal-coordinated tyrosinate to dioxygen as the dominant rate-limiting step.