Oxidation by mushroom tyrosinase of monophenols generating slightly unstable o-quinones

Abstract

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 < 10-3 s-1. The system evolves from an initial steady state, reached when t→0, 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.