Analysis and interpretation of the action mechanism of mushroom tyrosinaseon 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 la tter form is active on monophenols, while the former is not. However, the k inetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymati c 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, ty rosinase generates very unstable o-quinones, in which a process of cyclatio n and subsequent oxidation-reduction generates o-diphenol through non-enzym atic reactions. However, the release of o-diphenol through the action of th e enzyme on the monophenol contributes to the concentration of o-diphenol i n the first pseudo-steady-state [D-0](ss). Hence, the system reaches an ini tial 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 kineti c 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 tha t 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 oxidati on-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, in volving oxidation to o-quinone. The non-enzymatic evolution of the o-quinon e 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. Al l rights reserved.