Catalytic mechanism of the tyrosinase reaction toward the Tyr98 residue in the caddie protein
Catalytic mechanism of the tyrosinase reaction toward the Tyr98 residue in the caddie protein
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DOI：
10.1371/journal.pbio.3000077
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发表时间：
2018-12-01
期刊：
影响因子：
9.8
通讯作者：
Sugiyama, Masanori
中科院分区：
文献类型：
--
作者：
Matoba, Yasuyuki;Kihara, Shogo;Sugiyama, Masanori
酪氨酸酶（EC 1.14.18.1）是一种含铜的单加氧酶，催化苯酚转化为相应的邻醌。链霉菌酪氨酸酶与“球童”蛋白质形成复合物，促进两个铜离子转运到活性中心。在我们以前的研究中，已经发现容纳在酪氨酸酶活性中心口袋中的caddie蛋白中的Tyr（98）残基通过形成μ-η（2）：η（2）-过氧-二铜（II）和Cu（II）-多巴-半醌中间体而转化为活性醌。到目前为止，尽管基于晶体学分析、低分子量模型和计算机模拟对酪氨酸酶反应进行了广泛的研究，但仍无法在原子水平上阐明催化机制。为了使酪氨酸酶的催化机制更清楚，在本研究中，在非常高的分辨率（1.16-1.70 A）下测定了低温捕获的晶体结构。这些结构表明酪氨酸酶反应中存在一个尚未发现的重要步骤：即，羟基化反应是由Cu-A的运动引发的，其在形成μ-η（2）：η（2）-过氧二铜（II）核心后诱导铜配体的顺式到反式重排。通过重排，底物的羟基被置于赤道位置，允许Cu 2 O2氧化剂对芳环的亲电攻击。
Tyrosinase (EC 1.14.18.1), a copper-containing monooxygenase, catalyzes the conversion of phenol to the corresponding ortho-quinone. The Streptomyces tyrosinase is generated as a complex with a "caddie" protein that facilitates the transport of two copper ions into the active center. In our previous study, the Tyr(98) residue in the caddie protein, which is accommodated in the pocket of active center of tyrosinase, has been found to be converted to a reactive quinone through the formations of the mu-eta(2) :eta(2)-peroxo-dicopper(II) and Cu(II)-dopa-semiquinone intermediates. Until now-despite extensive studies for the tyrosinase reaction based on the crystallographic analysis, low-molecular-weight models, and computer simulations- the catalytic mechanism has been unable to be made clear at an atomic level. To make the catalytic mechanism of tyrosinase clear, in the present study, the cryo-trapped crystal structures were determined at very high resolutions (1.16-1.70 A). The structures suggest the existence of an important step for the tyrosinase reaction that has not yet been found: that is, the hydroxylation reaction is triggered by the movement of Cu-A, which induces the syn-to-anti rearrangement of the copper ligands after the formation of mu-eta(2):eta(2)-peroxodicopper(II) core. By the rearrangement, the hydroxyl group of the substrate is placed in an equatorial position, allowing the electrophilic attack to the aromatic ring by the Cu2O2 oxidant.