The Mechanism of Suicide-Inactivation of Tyrosinase: A Substrate Structure Investigation

Abstract

. The Mechanism of Suicide-Inactivation of Tyrosinase: A Substrate Structure Investigation. Tohoku J. Exp. Med., 2007, 212 (4), [341][342][343][344][345][346][347][348] 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 α -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.tyrosinase; suicideinactivation; catecholase; cresolase; α -oxidation; deprotonation © 2007 Tohoku University Medical Press When catechols are oxidised by tyrosinase there is a consistent anomaly in the oxygen stoichiometry. This phenomenon is amplified as the enzyme concentration is reduced. At low enzyme concentration the reaction ceases before either the substrate or the oxygen are depleted and further substrate supplementation or re-oxygenation have no effect. However, further enzyme addition re-initiates the reaction and the extent of further oxidation is a linear function of the amount of enzyme added. The effect is not due to a nonspecific influence of protein addition and re-initiation of oxidation is not observed when heat-inactivated tyrosinase is added to the reaction mixture. It is clear, therefore, that, during the oxidation of the substrate, a process occurs that

Oxidative discolouration in whole-head and cut lettuce: biochemical and environmental influences on a complex phenotype and potential breeding strategies to improve shelf-life

“…In addition to substrate activation, PPO from various sources, including lettuce has been shown to suffer from suicide inhibition, i.e. irreversible inhibition by a substrate analogue that occurs during the natural metabolic process (Chazarra et al 1999;Land et al 2007). In tyrosinase from Agaricus bisporus, these have been identified as catechols (o-diphenols) of various specific structures (Land et al 2007).…”

“…irreversible inhibition by a substrate analogue that occurs during the natural metabolic process (Chazarra et al 1999;Land et al 2007). In tyrosinase from Agaricus bisporus, these have been identified as catechols (o-diphenols) of various specific structures (Land et al 2007). Oxidation of diquinones produced at cut surfaces could theoretically lead to the production of small amounts of inhibitory catechols.…”

Studies of para-quinomethane formation during the tyrosinase-catalyzed oxidation of 4-alkylcatechols

“…In these experiments the catechol substrate (ca 0.4 mM) was added to the enzyme solution (300 units mL . 18 Thus, the rates of quinomethane formation are too slow to account for the inactivation. In a final experiment to support the conclusion that para-quinomethanes are not responsible for inactivation of tyrosinase we have shown that 4-methoxycatechol causes inactivation (k i 3.1 x 10 -2 s -1 ) even though it cannot form a quinomethane.…”

“…We have proposed that in addition to the normal catecholase oxidation that occurs via the complex 4 (Scheme 2A), a fraction of the catechol substrate presents itself as a cresolase substrate (Scheme 2B). 18 The alternative enzyme substrate complex 5 can undergo deprotonation and reductive elimination leading irreversibly to Cu(0) and inactive tyrosinase. …”

Mechanistic studies of tyrosinase suicide inactivation

“…Recently we proposed a mechanism that accounts for the copper loss during inactivation. 18 Briefly stated, this mechanism postulates that oxy-tyrosinase 1 sometimes binds catechol substrates in the oxygenase mode, i.e. as if they were phenols.…”