Slow-binding inhibition of mushroom (Agaricus bisporus) tyrosinase isoforms by tropolone

A kinetic study of the inhibition of mushroom tyrosinase by tropolone has been made. Three tyrosinase isoforms were used: two commercial tyrosinases from Fluka and Sigma (isoelectric points of 4.3 and 4.1, respectively) and one purified isoform from mushroom strain U1 (isoelectric point of 4.5). Tropolone is a slow-binding inhibitor of these mushroom tyrosinase isoforms. Increasing tropolone concentrations provoked a progressive decrease in both the initial velocity and the final (inhibited) steady-state rate in the progress curves of product accumulation. A rapid formation of an enzyme-inhibitor complex, which further undergoes a slow reversible reaction, could take place since the inhibition of the different isoforms was partially reversed by the addition of CuSO4. The kinetic parameters that described the inhibition by tropolone were evaluated by nonlinear regression fits. Incubation experiments of the different isoforms with tropolone demonstrated that this inhibitor only could bind to the "oxy" form of tyrosinase which justifies a mechanism previously proposed to explain the inhibition of tyrosinase by slow-binding inhibitors.