Kinetic study of monophenol and o-diphenol binding to oxytyrosinase

The complex reaction mechanism of tyrosinase involves three enzymatic forms, two overlapping catalytic cycles and a dead-end complex. The deoxytyrosinase form binds oxygen with a high degree of affinity, KsO2=46.6±2.4 μM. The mettyrosinase and oxytyrosinase forms bind monophenols and o-diphenols, although the former is inactive on monophenols. Analytical expressions for the catalytic and Michaelis constants of tyrosinase towards phenols and o-diphenols have been derived. Thus, the Michaelis constant of tyrosinase towards monophenols (KmM) and o-diphenols (KmD) are related with the catalytic constants for monophenols (kcatM) and o-diphenols (kcatD), respectively, and with the binding rate constants of the oxytyrosinase form with these substrates (k+4 and k+6, respectively), by means of the expressions KmM=kcatM/k+4 and KmD=kcatD/k+6. From these expressions, we calculate the values of the binding rate constant of oxytyrosinase to the substrates (monophenols and o-diphenols) for tyrosinases from different biological sources, and reveal that the o-diphenols bind more rapidly to oxytyrosinase than the monophenols. In addition, a new kinetic constant KmD(M)=kcatM/2k6 (the Michaelis constant for o-diphenol in the monophenolase activity), is derived and determined. Thus, it has been shown that tyrosinase has apparently higher affinity towards o-diphenols in its monophenolase than in its diphenolase activity.