Muñoz-Muñoz Jose L, García-Molina Francisco, García-Ruiz Pedro A, Molina-Alarcón Milagros, Tudela Jose, García-Cánovas Francisco, Rodríguez-López Jose N
Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
Biochem J. 2008 Dec 15;416(3):431-40. doi: 10.1042/BJ20080892.
The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone against time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism in which the enzymatic form E(ox) (oxy-tyrosinase) is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. The rate constant of this reaction would be directly related to the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon C-1 (delta(1)) obtained by (13)C-NMR. Protonation of the peroxide would bring the copper atoms together and encourage the diaxial nucleophilic attack of the C-2 hydroxyl group, facilitating the co-planarity with the ring of the copper atoms and the concerted oxidation/reduction reaction, and giving rise to an o-quinone. The suicide inactivation would occur if the C-2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the co-planarity between the copper atom, the oxygen of the C-1 and the ring would only permit the oxidation/reduction reaction on one copper atom, giving rise to copper(0), hydrogen peroxide and an o-quinone, which would be released, thus inactivating the enzyme.
酪氨酸酶作用于其底物的自杀失活机制已得到研究。在过渡阶段对所提出机制的动力学分析给出了邻醌浓度随时间变化的明确解析表达式。底物的电子、空间和疏水效应影响酶促反应，使催化速度提高三个数量级，失活程度提高一个数量级。为解释自杀失活现象，我们提出一种机制，即酶形式E(ox)（氧合酪氨酸酶）负责这种失活。关键步骤可能是C-1羟基的质子转移至过氧化物，过氧化物在此充当广义碱。另一个关键步骤可能是邻二酚对铜原子的轴向进攻。该反应的速率常数将直接与C-1羟基亲核进攻的强度相关，而这又取决于通过(13)C-NMR获得的碳C-1的化学位移（δ(1)）。过氧化物的质子化会使铜原子靠近，并促使C-2羟基进行双轴向亲核进攻，促进与铜原子环的共平面性以及协同氧化/还原反应，从而生成邻醌。如果C-2羟基将质子转移至质子化的过氧化物，自杀失活就会发生，此时过氧化物会再次充当广义碱。在这种情况下，铜原子、C-1的氧和环之间的共平面性仅允许一个铜原子发生氧化/还原反应，生成铜(0)、过氧化氢和一个将被释放的邻醌，从而使酶失活。
