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(O2) = 46.6 +/- 2.4 mu M. The mettyrosinase and oxytyrosinase forms I 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 m.1 cat 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) m cat 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 m cat 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)/2k(6) (the Michaelis constant for o-diphenol in the monophenolase activity), is m cat 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. (c) 2004 Elsevier B.V. All rights reserved.