Muñoz-Muñoz Jose Luis, Garcia-Molina Francisco, Garcia-Ruiz Pedro Antonio, Varon Ramon, Tudela Jose, Rodriguez-Lopez Jose N, Garcia-Canovas Francisco
Departamento de Bioquímica y Biología Molecular-A, Universidad de Murcia, Espinardo, Murcia, Spain.
Biochim Biophys Acta. 2011 Dec;1814(12):1974-83. doi: 10.1016/j.bbapap.2011.07.015. Epub 2011 Jul 22.
The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643-650). In the case of o-aminophenols, the hydroxyl group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are of the same order of magnitude than for monophenols, which suggests that the monophenols bind better (higher binding constant) to the enzyme to facilitate the π-π interactions between the aromatic ring and a possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis constants are low, the values of the catalytic constants being lower than those of the corresponding o-diphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding constant) to the enzyme.
研究了酪氨酸酶以邻氨基酚和芳香胺为底物时的动力学。芳香单胺和邻二胺的催化常数都很低，这些结果与我们之前提出的机制一致，即在该机制中，慢步骤是氧合酪氨酸酶中一个羟基将质子转移至过氧化物上（费诺尔等人，《生物化学杂志》380卷（2004年）643 - 650页）。对于邻氨基酚，羟基在质子转移过程中间接发挥协同作用，因此酪氨酸酶作用于这些化合物时的催化常数更高。对于芳香单胺，米氏常数与单酚的米氏常数处于同一数量级，这表明单酚与酶的结合更好（结合常数更高），以促进芳香环与活性位点中可能存在的组氨酸之间的π - π相互作用。对于芳香邻二胺，催化常数和米氏常数都很低，催化常数的值低于相应邻二酚的催化常数。芳香邻二胺的米氏常数的值略低于其相应邻二酚的米氏常数，这证实了芳香邻二胺与酶的结合较差（结合常数较低）。
