Detection of the reaction intermediates catalyzed by a copper amine oxidase

Misumi Kataoka,Katsuyuki Tanizawa,Ayuko Tominaga,Toshihide Okajima,Hiroshi Yamaguchi,Masayuki Otsu,Hiroko Oya

doi:10.1107/s0909049510034989

Misumi Kataoka, Katsuyuki Tanizawa + Show 5 more

Open Access

https://doi.org/10.1107/s0909049510034989

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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.

Highlights

To elucidate the mechanism of an enzyme reaction, it is important to characterize the enzymatic properties and determine the enzyme’s function
We have studied the CAO phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter globiformis (AGAO)
436 and 466 nm, arose with the passage of time; peaks at 60 min, similar to 120 min, were 436 and 466 nm, which were consistent with those of TPQsq. These results suggested that AGAO in crystals reacted with PEA during the time course from 2 to 60 min

Summary

Introduction

To elucidate the mechanism of an enzyme reaction, it is important to characterize the enzymatic properties and determine the enzyme’s function. Studies of reaction mechanisms have usually used biochemical and physicochemical methods such as spectroscopy. Using these techniques it is possible to determine the reaction mechanism by characterizing the amino acids that directly participate in the chemical reaction. Copper amine oxidases (EC 1.4.3.6) (CAOs) catalyze the oxidative deamination of primary amines to their corresponding aldehydes with the concomitant production of hydrogen peroxide and ammonia. In their active sites these enzymes contain a copper ion and a redox-active organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (topaquinone, TPQox), which is a quinone cofactor that is covalently linked to the polypeptide chain. TPQox is produced by the post-translational modification of the conserved precursor tyrosine residue in the presence of copper ion and molecular oxygen

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