title: Detection of the reaction intermediates catalyzed by a copper amine oxidase.
abstract: To reveal the chemical changes and geometry changes of active-site residues that cooperate with a reaction is important for understanding the functional mechanism of proteins. Consecutive temporal analyses of enzyme structures have been performed during reactions to clarify structure-based reaction mechanisms. Phenylethylamine oxidase from Arthrobacter globiformis (AGAO) contains a copper ion and topaquinone (TPQ(ox)). The catalytic reaction of AGAO catalyzes oxidative deaminations of phenylethylamine and consists of reductive and oxidative half-reactions. In the reduction step, TPQ(ox) reacts with a phenylethylamine (PEA) substrate giving rise to a topasemiquinone (TPQ(sq)) formed Schiff-base and produces phenylacetaldehyde. To elucidate the mechanism of the reductive half-reaction, an attempt was made to trap the reaction intermediates in order to analyze their structures. The reaction proceeded within the crystals when AGAO crystals were soaked in a PEA solution and freeze-trapped in liquid nitrogen. The reaction stage of each crystal was confirmed by single-crystal microspectrometry, before X-ray diffraction measurements were made of four reaction intermediates. The structure at 15 min after the onset of the reaction was analyzed at atomic resolution, and it was shown that TPQ(ox) and some residues in the substrate channel were alternated via catalytic reductive half-reactions.
authors.affiliation: School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
authors.forename: Misumi
authors.initials: M
authors.lastname: Kataoka
authors.forename: Hiroko
authors.initials: H
authors.lastname: Oya
authors.forename: Ayuko
authors.initials: A
authors.lastname: Tominaga
authors.forename: Masayuki
authors.initials: M
authors.lastname: Otsu
authors.forename: Toshihide
authors.initials: T
authors.lastname: Okajima
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Tanizawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamaguchi
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000378:metabolism*; D001173:Arthrobacter / Q000201:enzymology; D002384:Catalysis; D018360:Crystallography, X-Ray; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010627:Phenethylamines / Q000737:chemistry / Q000378:metabolism; D012545:Schiff Bases; D014961:X-Ray Diffraction
chemical_list: D010627:Phenethylamines; D012545:Schiff Bases; C029261:phenethylamine; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; C086534:phenylethylamine oxidase; D006631:Amine Oxidase (Copper-Containing)