School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
J Synchrotron Radiat. 2011 Jan;18(1):58-61. doi: 10.1107/S0909049510034989. Epub 2010 Nov 5.
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.
揭示与反应协同作用的活性位点残基的化学变化和几何变化对于理解蛋白质的功能机制很重要。在反应过程中连续进行酶结构的时间分析，以阐明基于结构的反应机制。来自球形节杆菌（Arthrobacter globiformis）的苯乙胺氧化酶（AGAO）含有铜离子和托普醌（TPQ(ox)）。AGAO 的催化反应催化苯乙胺的氧化脱氨，由还原和氧化半反应组成。在还原步骤中，TPQ(ox)与苯乙胺（PEA）底物反应，生成形成席夫碱的半醌（TPQ(sq))，并产生苯乙醛。为了阐明还原半反应的机制，尝试捕获反应中间体以分析其结构。当 AGAO 晶体在 PEA 溶液中浸泡并在液氮中冷冻捕获时，反应在晶体内部进行。在进行 X 射线衍射测量之前，通过单晶微光谱法确认每个晶体的反应阶段，对四个反应中间体进行了测量。在反应开始 15 分钟后，对原子分辨率进行了分析，结果表明，TPQ(ox)和底物通道中的一些残基通过催化还原半反应交替。
