Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones
Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones
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DOI：
10.1016/s0167-4838(01)00207-2
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发表时间：
2001-07-09
期刊：
影响因子：
--
通讯作者：
Tudela, J
中科院分区：
文献类型：
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作者：
Fenoll, LG;Rodríguez-López, JN;Tudela, J
酪氨酸酶可以作用于单酚，因为它是以酶的天然形式存在的MET-(E-m)和氧基酪氨酸酶(E-OX)的混合物。后一种形式对单酚具有活性，而前一种形式不起作用。然而，动力学是复杂的，因为单酚可以与这两种酶结合。这种情况变得更加复杂，因为酶反应的产物邻苯二酚是不稳定的，并继续在介质中生成邻二酚。在底物如L酪氨酸的情况下，酪氨酸酶产生非常不稳定的邻苯二酚，在这个过程中，环化和随后的氧化还原过程通过非酶反应产生邻二酚。然而，通过酶对单酚的作用释放邻二酚，导致邻二酚在第一个拟稳定状态[D-0](Ss)中的浓度。因此，体系在t->；0时达到初始拟稳态，并经历过渡阶段(滞后期)，直到介质中邻二酚浓度达到最终稳态浓度[D-f](Ss)时达到最终稳态。这些结果可以通过考虑酶的动力学和结构机制来解释。在这种情况下，酪氨酸酶将单酚羟基化为邻二酚，生成中间体EMD，该中间体可能氧化邻二酚或将其直接释放到介质中。推测E-OX与单酚作用生成的中间体EMD与活性中心的铜原子之间存在轴向和赤道键。由于轨道不是共面的，不可能发生协同氧化还原反应。相反，一个键，可能是C-4的键被打破，以实现共面，产生更不稳定的中间体，然后将邻二酚释放到介质中或将其向轴向团聚，包括氧化成邻苯二酚。邻苯二酚的非酶演化将产生在滞后期后达到最终稳定状态所必需的邻二酚([D-f](Ss))。(C)2001 Elsevier Science B.V.保留所有权利。
Tyrosinase can act on monophenols because of the mixture of met- (E-m) and oxy-tyrosinase (E-ox) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D-0](ss). Hence, the system reaches an initial pseudo-steady state when t --> 0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D-f](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, EmD, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E-ox on monophenols, EmD, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D-f](ss)) necessary for the final steady state to be reached after the lag period. (C) 2001 Elsevier Science B.V. All rights reserved.