Tohoku J. Exp. Med., 2007, 212Tyrosinase, 341-348 Suicide-Inactivation Mechanism 341 The Mechanism of Suicide-Inactivation of Tyrosinase: A Substrate Structure Investigation 1 1 2 EDWARD J. LAND, CHRISTOPHER A. RAMSDEN and PATRICK A. RILEY 1Lennard-Jones Laboratories, School of Physical and Geographical Sciences, Keele University, Staffordshire, U.K. 2Totteridge Institute for Advanced Studies, London, U.K. LAND, E.J., RAMSDEN, C.A. and RILEY, P.A. The Mechanism of Suicide-Inactivation of Tyrosinase: A Substrate Structure Investigation. Tohoku J. Exp. Med., 2007, 212 (4), 341-348 ── 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 inacti- vation which occurs during the oxidation of catechols. Although the corresponding cate- chols are formed during tyrosinase oxidation of monophenols, inactivation in the presence of monophenolic substrates is minimal. Previous studies have established the kinetic fea- tures of the inactivation reaction which is first-order in respect of the enzyme concentra- tion. 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 inactiva- tion 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 adja- cent group. On the basis of our experimental data we believe that a similar mechanism may account for the inhibitory action of resorcinols. ────── tyrosinase; suicide- inactivation; catecholase; cresolase; α-oxidation; deprotonation © 2007 Tohoku University Medical Press When catechols are oxidised by tyrosinase re-initiates the reaction and the extent of further there is a consistent anomaly in the oxygen stoi- oxidation is a linear function of the amount of chiometry. This phenomenon is amplified as the enzyme added. The effect is not due to a non- enzyme concentration is reduced. At low enzyme specific influence of protein addition and concentration the reaction ceases before either the re-initiation of oxidation is not observed when substrate or the oxygen are depleted and further heat-inactivated tyrosinase is added to the reac- substrate supplementation or re-oxygenation have tion mixture. It is clear, therefore, that, during the no effect. However, further enzyme addition oxidation of the substrate, a process occurs that Received May 2, 2007; revision accepted for publication May 30, 2007. Correspondence: Prof. P.A. Riley, Totteridge Institute for Advanced Studies, The Grange, Grange Avenue, London N20 8AB, U.K. e-mail: [email protected] 341 342 E.J. Land et al. Tyrosinase Suicide-Inactivation Mechanism 343 inactivates tyrosinase. This phenomenon, often catechol substrate as a cresol, i.e. a “cresolase” referred to as “suicide inactivation”, is a charac- presentation as opposed to a “catecholase” pre- teristic of several enzymes (Walsh 1984) and has sentation (Mason 1955). This leads to the cate- long been recognised as a feature of both plant chol being oxidised to form a product able to and animal tyrosinases (Nelson and Dawson undergo deprotonation and reductive elimination 1944). of an ortho-quinone which results in inactivation The biological significance of the reaction- of the enzyme by formation of copper(0) at the inactivation may be as a limitation of the activity active site. A similar mechanism may account for of an enzyme generating potentially cytotoxic the inhibitory properties of resorcinols. oxidation products. It has been the subject of many studies (Asimov and Dawson 1950; MATERIALS AND METHODS Ingraham et al. 1952; Tomita and Seiji 1977; Seiji Chemicals et al. 1978; Tomita et al. 1980; Lerch 1983; The reagents used in this study were purchased from Miranda and Botti 1983; Waley 1985; Garcia- Sigma-Aldrich, Poole, Dorset, UK. Tyrosinase (from Canovas et al. 1987; Tudela et al. 1988; Haghbeen Agaricus bisporus) was made up at a concentration of et al. 2004; Garcia-Molina et al. 2005), but the 300 (Sigma) units per ml in 0.1 M phosphate buffer (pH details of the mechanism of the inactivation have 7.4), frozen in aliquots of 5 ml, and stored at –20°C. remained unclear. Solutions of substrates were freshly prepared in glass distilled water. The agents tested included a range of Two general mechanistic proposals to potential tyrosinase substrates shown in Table 1. The account for the inactivation have been advanced: following compounds were not commercially available (1) an attack by the ortho-quinone product of oxi- and were prepared by the literature methods cited: 4,6-di- dation on a sensitive nucleophilic group vicinal to methylresorcinol (Cram and Cranz 1950); 4-fluorocate- the active site (Ingraham et al. 1952), and (2) a chol (Corse and Ingraham 1951); (3-hydroxyphenyl) free radical attack on the active site by reactive acetonitrile (Salkowski 1884). 4-Methoxycatechol was oxygen species generated during the catalytic oxi- prepared by Mr. C.J. Cooksey (University College dation (Seiji et al. 1978). However, experiments London) and 3,6-dimethylcatechol was provided by in which ortho-quinone binding was prevented Professor Marco d’Ischia (University of Naples). failed to influence inactivation and attempts to protect the enzyme with radical scavengers Oximetry proved unsuccessful (Tomita et al. 1980; Dietler Experiments were conducted at 30°C using an and Lerch 1982). apparatus consisting of a quartz cuvette (3.65 mls capa- In this study we examined the reaction-inac- city) adapted to hold a Clark-type oxygen electrode, tivation of mushroom tyrosinase using a range of as described previously (Cooksey et al. 1997). substrates and have examined both the kinetics of Spectrophotometric data were recorded using a Hewlett- the process and structural aspects of the substrate Packard diode-array spectrophotometer (Model 8452A) specificity. From these data we have derived a and the oxygen uptake monitored using a Yellow Springs plausible mechanism of suicide inactivation. A Instruments (Model 5300) polarimeter. Oxygen elec- trode tracings were converted to electronic form using kinetic model of catechol metabolism by compet- ScanIt software (Version 1.0, J. van Baten and R. Baur ing alternative pathways, one yielding the normal 2002). Kinetic analysis was conducted using the Origin ortho-quinone oxidation product and the other 61 program (OriginLab Co., Northampton, MA, USA). generating a product that inactivates the enzyme, Simulations were performed using an in-house computer closely simulates the kinetic features of the phe- model. Spectral changes were examined using the kinet- nomenon and exhibits a near-linear relationship ic mode of the UV-Vis Chemstation A0801(66) software between the extent of oxidation and the amount of (Agilent Technologies, Hannover, Germany). Unless added enzyme, as found experimentally. In this otherwise stated the assays were performed at pH 6.75 paper we propose that the inactivation mechanism with a substrate concentration of 820 μM and enzyme involves a presentation at the active site of the concentrations between 1-10 units/ml. The total oxygen 342 E.J. Land et al. Tyrosinase Suicide-Inactivation Mechanism 343 utilization was found to fit the equation: tive inactivation rate. The ratio k2/k1 represents the proportion of inactivating to catalytic reac- k1 tions and this varies between 1.4 and 10% for the Ut = E0 (1 – exp [-k2t]) compounds tested. Inactivation was confirmed by k2 zero residual activity (RA in Table 1) of the where Ut = oxygen utilised at time t; E0 = initial enzyme after cessation of oxygen uptake. amount of enzyme; k1 = oxidation rate; k2 = inactivation Residual activity was tested both for catecholase rate of the enzyme. From the total oxygen utilized (UT) (RAcat) and cresolase (RAcr) using 4-methylcate- the oxidation rate was derived as: k1 = UT.k2/E0. The chol and 4-methylphenol as substrates respective- standard test substrate was 4-methylcatechol which ly. exhibits a linear relationship between the total oxygen Of the benzenetriols, 1,3,5-benzenetriol utilization and the amount of enzyme (UT = 24.1 nano- (entry 15) was neither a primary substrate nor an 2 moles oxygen per unit, r = 0.9577). To test for residual inactivator. 1,2,4-Benzenetriol (entry 12) was catecholase activity (RAcat in Table 1) 4-methylcatechol shown to be a substrate for tyrosinase and seemed was added at the end of the oxygen uptake or after 5 to possess little inactivating activity, but the minutes if no oxygen uptake was observed and the sec- experiments had to be performed at pH 3.5 (indi- ondary oxygen utilization measured and expressed as a cated by note [a] in Table 1) to halt the rapid percentage of the control value. 4-Methylphenol was autoxidation of this compound, so direct compari- used as competitive cresolase substrate and for the esti- son with the data from other compounds is not mation of residual