Land Edward J, Ramsden Christopher A, Riley Patrick A
Lennard-Jones Laboratories, School of Physical and Geographical Sciences, Keele University, Staffordshire, UK.
Tohoku J Exp Med. 2007 Aug;212(4):341-8. doi: 10.1620/tjem.212.341.
Tyrosinase is a copper-containing mono-oxygenase, widely distributed in nature, able to catalyze the oxidation of both phenols and catechols to the corresponding ortho-quinones. Tyrosinase is characterised by a hitherto unexplained irreversible inactivation which occurs during the oxidation of catechols. Although the corresponding catechols are formed during tyrosinase oxidation of monophenols, inactivation in the presence of monophenolic substrates is minimal. Previous studies have established the kinetic features of the inactivation reaction which is first-order in respect of the enzyme concentration. The inactivation reaction exhibits the same pH-profile and saturation properties as the oxidation reaction, classing the process as a mechanism-based suicide inactivation. The recent elucidation of the crystallographic structure of tyrosinase has stimulated a new approach to this long-standing enigma. Here we report the results of an investigation of the tyrosinase-catalysed oxidation of a range of hydroxybenzenes which establish the structural requirements associated with inactivation. We present evidence for an inactivation mechanism based on catechol hydroxylation, with loss of one of the copper atoms at the active site. The inactivation mechanism involves two linked processes occurring in situ: (a) catechol presentation resulting in alpha-oxidation, and (b) deprotonation of an adjacent group. On the basis of our experimental data we believe that a similar mechanism may account for the inhibitory action of resorcinols.
酪氨酸酶是一种含铜的单加氧酶，广泛分布于自然界，能够催化酚类和儿茶酚类氧化为相应的邻醌。酪氨酸酶的特点是在儿茶酚氧化过程中会发生一种迄今无法解释的不可逆失活。尽管在单酚的酪氨酸酶氧化过程中会形成相应的儿茶酚，但在单酚底物存在下的失活程度极小。以往的研究已经确定了失活反应的动力学特征，该反应对酶浓度而言是一级反应。失活反应与氧化反应表现出相同的pH曲线和饱和特性，将该过程归类为基于机制的自杀失活。最近对酪氨酸酶晶体结构的阐明激发了对这一长期谜团的新研究方法。在此，我们报告了对一系列羟基苯的酪氨酸酶催化氧化的研究结果，这些结果确定了与失活相关的结构要求。我们提供了一种基于儿茶酚羟基化的失活机制的证据，即活性位点的一个铜原子丢失。失活机制涉及原位发生的两个相关过程：(a)儿茶酚呈现导致α-氧化，以及(b)相邻基团的去质子化。基于我们的实验数据，我们认为类似的机制可能解释间苯二酚的抑制作用。
