Matoba Yasuyuki, Oda Kosuke, Muraki Yoshimi, Masuda Taro
Faculty of Pharmacy, Yasuda Women's University, Yasuhigashi 6-13-1, Asaminami-ku, Hiroshima, 731-0153, Japan.
Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Int J Biol Macromol. 2021 Jul 31;183:1861-1870. doi: 10.1016/j.ijbiomac.2021.05.206. Epub 2021 Jun 2.
Tyrosinase (Ty) and catechol oxidase (CO) are members of type-3 copper enzymes. While Ty catalyzes both phenolase and catecholase reactions, CO catalyzes only the latter reaction. In the present study, Ty was found to catalyze the catecholase reaction, but hardly the phenolase reaction in the presence of the metallochaperon called "caddie protein (Cad)". The ability of the substrates to dissociate the motif shielding the active-site pocket seems to contribute critically to the substrate specificity of Ty. In addition, a mutation at the N191 residue, which forms a hydrogen bond with a water molecule near the active center, decreased the inherent ratio of phenolase versus catecholase activity. Unlike the wild-type complex, reaction intermediates were not observed when the catalytic reaction toward the Y98 residue of Cad was progressed in the crystalline state. The increased basicity of the water molecule may be necessary to inhibit the proton transfer from the conjugate acid to a hydroxide ion bridging the two copper ions. The deprotonation of the substrate hydroxyl by the bridging hydroxide seems to be significant for the efficient catalytic cycle of the phenolase reaction.
酪氨酸酶（Ty）和儿茶酚氧化酶（CO）属于3型铜酶。虽然Ty催化酚酶和儿茶酚酶反应，但CO仅催化后者反应。在本研究中，发现Ty在存在称为“携带蛋白（Cad）”的金属伴侣蛋白的情况下催化儿茶酚酶反应，但几乎不催化酚酶反应。底物解离屏蔽活性位点口袋的基序的能力似乎对Ty的底物特异性起关键作用。此外，在与活性中心附近的水分子形成氢键的N191残基处的突变降低了酚酶与儿茶酚酶活性的固有比率。与野生型复合物不同，当在结晶状态下对Cad的Y98残基进行催化反应时，未观察到反应中间体。水分子碱性的增加可能是抑制质子从共轭酸转移到桥接两个铜离子的氢氧根离子所必需的。桥接氢氧根对底物羟基的去质子化似乎对酚酶反应的有效催化循环很重要。
