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

A kinetic study of the inhibition of mushroom tyrosinase by tropolone has b een made. Three tyrosinase isoforms were used: two commercial tyrosinases f rom Fluka and Sigma (isoelectric points of 4.3 and 4.1, respectively) and o ne purified isoform from mushroom strain U1 (isoelectric point of 4.5). Tro polone is a slow-binding inhibitor of these mushroom tyrosinase isoforms. I ncreasing tropolone concentrations provoked a progressive decrease in both the initial velocity and the final (inhibited) steady-state rate in the pro gress curves of product accumulation. A rapid formation of an enzyme-inhibi tor complex, which further undergoes a slow reversible reaction, could take place since the inhibition of the different isoforms was partially reverse d by the addition of CuSO4. The kinetic parameters that described the inhib ition 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.