Matoba Yasuyuki, Kihara Shogo, Muraki Yoshimi, Bando Naohiko, Yoshitsu Hironari, Kuroda Teruo, Sakaguchi Miyuki, Kayama Kure'e, Tai Hulin, Hirota Shun, Ogura Takashi, Sugiyama Masanori
Graduate School of Biomedical & Health Sciences, Hiroshima University , Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Picobiology Institute, Graduate School of Life Science, University of Hyogo, RSC-UH Leading Program Center , Koto 1-1-1, Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
Biochemistry. 2017 Oct 17;56(41):5593-5603. doi: 10.1021/acs.biochem.7b00635. Epub 2017 Sep 27.
Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding ortho-quinone. The enzyme from the genus Streptomyces is generated as a complex with a "caddie" protein that assists the transport of two copper ions into the active center. In this complex, the Tyr residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr residue was converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated that the conversion occurred through the formations of μ-η:η-peroxo-dicopper(II) and Cu(II)-semiquinone. Electron paramagnetic resonance analysis under anaerobic conditions and Fourier transform infrared spectroscopic analysis using CO as a structural probe under anaerobic conditions indicated that the copper transportation process to the active center is a reversible event in the tyrosinase/caddie complex. Aggregation of the caddie protein, which is triggered by the conversion of the Tyr residue to dopaquinone, may ensure the generation of fully activated tyrosinase.
酪氨酸酶（EC 1.14.18.1）在活性中心含有两个铜离子，催化黑色素生成的限速反应，即苯酚转化为相应的邻醌。链霉菌属的这种酶是与一种“携带蛋白”形成复合物产生的，该携带蛋白协助将两个铜离子转运到活性中心。在这个复合物中，发现携带蛋白中的酪氨酸残基位于酪氨酸酶活性中心的口袋中，其方式可能类似于作为酪氨酸酶真正底物的L-酪氨酸。在生理条件下，向复合物中添加铜离子会根据携带蛋白的聚集从复合物中释放酪氨酸酶。发现在有氧条件下添加还原剂会加速与铜结合的酪氨酸酶的释放。质谱分析表明酪氨酸残基转化为反应性醌，共振拉曼光谱分析表明这种转化是通过形成μ-η:η-过氧-二铜（II）和铜（II）-半醌发生的。厌氧条件下的电子顺磁共振分析和使用一氧化碳作为结构探针的厌氧条件下的傅里叶变换红外光谱分析表明，在酪氨酸酶/携带蛋白复合物中，铜向活性中心的转运过程是一个可逆事件。携带蛋白的聚集由酪氨酸残基向多巴醌的转化引发，这可能确保产生完全活化的酪氨酸酶。
