Tyrosinase can act on monophenols because of the mixture of mettyrosinase (E m ) and oxytyrosinase (E ox ) that exists in the native form of the enzyme. The latter form is active on monophenols although the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex as the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as 4-methoxyphenol, 4-ethoxyphenol and 4-tert-butylphenol, tyrosinase generates o-quinones which become unstable with small constants of approximately , 10 23 s 21 . The system evolves from an initial steady state, reached when t30, through a transition state towards a final steady state, which is never reached because the substrate is largely consumed. The mechanisms proposed to explain the enzyme's action can be differentiated by the kinetics of the first steady state. The results suggest that tyrosinase hydroxylates monophenols to o-diphenols, generating an intermediate E m -diphenol in the process, which may oxidize the o-diphenol or release it directly into the medium. In the case of o-quinone formation, its slow instability generates o-diphenol which activates the enzymatic system yielding parabolic time recordings.Keywords: enzyme kinetics; monophenol; mushroom; o-diphenol; o-quinone; tyrosinase.Tyrosinase is a copper-enzyme [1,2] widely distributed throughout the phylogenetic scale [3,4] that catalyses the hydroxylation of monophenols to o-diphenols and the oxidation of the latter to o-quinones using molecular oxygen [5]. The enzyme is responsible for skin, eye, inner ear and hair melanization [6,7], and browning in fruits and vegetables [8±10]. Its action on its physiological substrate, l-tyrosine, has been studied widely and its kinetic has been largely interpreted [11±14]. Studies have also been made of its action on monophenols such as 4-hydroxyanisole (4HA), 4-ethoxyphenol (4EP) and 4-tert-butylphenol (4TBP), to produce fairly stable o-quinones [15±20]. In 1987, our group [12] proposed a kinetic mechanism based on structural aspects developed by Solomon [21] to explain the enzyme's action (Scheme 1a). Briefly, the oxy form of the enzyme (E ox ) starts the turnover by acting on the monophenol (M), which is hydroxylated to the mettyrosine± diphenol intermediate (E m D). At this point, the enzyme can oxidize D to o-quinone (Q), producing the desoxy form of the enzyme (E d ), or release D to form the met (E m ) form, which binds with M to produce the inactive form, E m M. If Q is very unstable, as occurs in the case of o-dopaquinone, D is recycled to the medium through intramolecular cyclization and further redox steps [12]. This would involve the transformation of the E m form of the enzyme (which is inactive towards M) into the E ox form (which is active towards M) giving rise to the lag time, which is a characteristic of this activity (Scheme 1a 1 b) [12±14]. In such cases the system would re...
