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, K s O 2 = 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 ( K m M ) and o-diphenols ( K m D ) are related with the catalytic constants for monophenols ( k cat M ) and o-diphenols ( k cat D ) , 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 K m M = k cat M / k + 4 and K m D = k cat D / 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 K m D ( M ) = k cat M / 2 k 6 (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.