Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones

Tyrosinase can act on monophenols because of the mixture of met- (E-m) and oxy-tyrosinase (E-ox) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since 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 L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D-0](ss). Hence, the system reaches an initial pseudo-steady state when t --> 0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D-f](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, EmD, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E-ox on monophenols, EmD, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D-f](ss)) necessary for the final steady state to be reached after the lag period. (C) 2001 Elsevier Science B.V. All rights reserved.