Mushroom Tyrosinase: Catalase Activity, Inhibition, and Suicide Inactivation FRANCISCO GARCI ÄA-MOLINA, † ALEXANDER N. P. HINER, † LORENA G. FENOLL, † JOSE Ä N. RODRI ÄGUEZ-LOPEZ, † PEDRO A. GARCI ÄA-RUIZ, ‡ FRANCISCO GARCI ÄA-CA Ä NOVAS,* ,† AND JOSE Ä TUDELA † GENZ: Grupo de Investigacio ´n de Enzimologı ´a, Departamento de Bioquı ´mica y Biologı ´a Molceular-A, Facultad de Biologı ´a, and Departamento de Quı ´mica Orga ´nica, Facultad de Quı ´mica, Universidad de Murcia, E-0100 Espinardo, Murcia, Spain Mushroom tyrosinase exhibits catalase activity with hydrogen peroxide (H 2 O 2 ) as substrate. In the absence of a one-electron donor substrate, H 2 O 2 is able to act as both oxidizing and reducing substrate. The kinetic parameters V max and K m that characterize the reaction were determined from the initial rates of oxygen gas production (V 0 O 2) under anaerobic conditions. The reaction can start from either of the two enzyme species present under anaerobic conditions: met-tyrosinase (E m ) and deoxy-tyrosinase (E d ). Thus, a molecule of H 2 O 2 can reduce E m to E d via the formation of oxy-tyrosinase (E ox ) (E m + H 2 O 2 h E ox ), E ox releases oxygen into the medium and is transformed into E d , which upon binding another molecule of H 2 O 2 is oxidized to E m . The effect of pH and the action of inhibitors have also been studied. Catalase activity is favored by increased pH, with an optimum at pH ) 6.4. Inhibitors that are analogues of o-diphenol, binding to the active site coppers diaxially, do not inhibit catalase activity but do reduce diphenolase activity. However, chloride, which binds in the equatorial orientation to the protonated enzyme (E m H), inhibits both catalase and diphenolase activities. Suicide inactivation of the enzyme by H 2 O 2 has been demonstrated. A kinetic mechanism that is supported by the experimental results is presented and discussed. KEYWORDS: Tyrosinase; mushroom; catalase; inhibition; suicide inactivation INTRODUCTION Tyrosinase (EC 1.14.18.1), often also referred to as poly- phenol oxidase (PPO), is a copper-containing mono-oxygenase, present in a diverse range of organisms, that is responsible for melanization in animals and the enzymatic browning of fruit. The enzyme catalyzes two distinct reactions involving molecular oxygen: the hydroxylation of monophenols to o-diphenols and the oxidation of the latter to o-quinones (1-3). Chemical and spectroscopic studies of tyrosinase have shown that its binuclear copper active site can be prepared in several forms: met, deoxy, and oxy (4-6). The oxygenated form, oxy- tyrosinase (E ox ), is capable of acting on both monophenols and o-diphenols. met-tyrosinase (E m ) does not act on monophenols but can be converted to E ox by the addition of hydrogen peroxide (H 2 O 2 )(4-6). deoxy-Tyrosinase (E d ) will bind molecular oxygen (O 2 ) to form E ox (6). Most of the enzyme in a freshly prepared sample (resting tyrosinase) is in the E m form unable to bind O 2 ; only a small fraction is present as E ox , this being necessary to initiate catalysis with monophenols (7). Recently, a mechanism has been published for the catalase activity of a catechol oxidase (CAO) isoenzyme from Ipomoea batatas (sweet potato) (8). These ubiquitous plant enzymes lack monooxygenase activity in contrast to tyrosinase (9). CAO and tyrosinase, together with hemocyanin, the O 2 transport protein of many arthropods, possess an antiferromagnetically coupled dinuclear copper center in the met and oxy states. These so- called type-3 copper centers bind and/or activate O 2 (10, 11) and share similar spectroscopic features (12). Catalase activity has been described in several hemocyanins (13-15). In this case, the proposed mechanism is initiated by the binding of the first H 2 O 2 molecule to the reduced (Cu + Cu + ) deoxy state of the copper oxidizing it to Cu 2+ Cu 2+ (met-hemocyanin). The second H 2 O 2 now enters the active site and forms the oxy state with the dinuclear copper. The H 2 O 2 is then oxidized to O 2 , and the copper returns to the Cu + Cu + state. The catalase cycle has not been described in the case of tyrosinase, although the following reaction was described some time ago (4-6). In the case of CAO, catalase activity has been described in one isoenzyme (8). The proposed mechanism involves binding of the first H 2 O 2 to the met form, displacing a hydroxo group bound * Author to whom correspondence should be addressed [fax +34 968 363963; e-mail canovasf@um.es]. † Facultad de Biologı ´a. ‡ Facultad de Quı ´mica. E m + H 2 O 2 h E ox h E d + O 2 (1) 3702 J. Agric. Food Chem. 2005, 53, 3702-3709 10.1021/jf048340h CCC: $30.25 © 2005 American Chemical Society Published on Web 04/05/2005