Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions.
Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions.
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卤离子探讨球形节杆菌铜胺氧化酶的催化机制。
DOI：
10.1074/jbc.m115.662726
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
T.
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作者：
Murakawa;T.;Hamaguchi;A.;Nakanishi;S.;Kataoka;M.;Nakai;T.;Kawano;Y.;Yamaguchi;H.;Hayashi;H.;Tanizawa;K.;and Okajima;T.
铜胺氧化酶的催化反应通过乒乓机理进行，包括两个半反应。在最初的半反应中，底物胺通过分子内电子转移到活性中心铜，将Tyr衍生的辅因子Topa Quone(TPQ)还原为氨基间苯二酚(TPQamr)，该形式与半醌自由基(TPQsq)保持平衡。我们分析了球状节杆菌铜胺氧化酶晶体中的这种还原半反应。以胺为底物的厌氧浸泡使晶体的平衡向“铜上”构象转移，其中4-羟基轴向连接到铜中心，铜中心可能被还原为铜(I)。当晶体在卤化物离子存在下与底物浸泡时，晶体中的平衡向非铜构象的TPQamr移动。卤化物离子分别对胺底物和氧气起非竞争性和非竞争性的抑制作用。卤化物离子被结合到铜中心的轴向位置，从而阻止了TPQamr采用铜上的构象。此外，在粘原(甘油)存在下的瞬时动力学分析表明，只有TPQamr/TPQq相互转化过程中的速率常数受到粘原的显著影响，这可能扰乱了构象的变化。这些发现明确地证明了TPQ在还原半反应过程中发生了很大的构象变化。
The catalytic reaction of copper amine oxidase proceeds through a ping-pong mechanism comprising two half-reactions. In the initial half-reaction, the substrate amine reduces the Tyr-derived cofactor, topa quinone (TPQ), to an aminoresorcinol form (TPQamr) that is in equilibrium with a semiquinone radical (TPQsq) via an intramolecular electron transfer to the active-site copper. We have analyzed this reductive half-reaction in crystals of the copper amine oxidase fromArthrobacter globiformis. Anerobic soaking of the crystals with an amine substrate shifted the equilibrium toward TPQsqin an “on-copper” conformation, in which the 4-OH group ligated axially to the copper center, which was probably reduced to Cu(I). When the crystals were soaked with substrate in the presence of halide ions, which act as uncompetitive and noncompetitive inhibitors with respect to the amine substrate and dioxygen, respectively, the equilibrium in the crystals shifted toward the “off-copper” conformation of TPQamr. The halide ion was bound to the axial position of the copper center, thereby preventing TPQamrfrom adopting the on-copper conformation. Furthermore, transient kinetic analyses in the presence of viscogen (glycerol) revealed that only the rate constant in the step of TPQamr/TPQsqinterconversion is markedly affected by the viscogen, which probably perturbs the conformational change. These findings unequivocally demonstrate that TPQ undergoes large conformational changes during the reductive half-reaction.