TYROSINASE AUTOACTIVATION AND THE PROBLEM OF THE LAG PERIOD

Evidence is presented for the binding of the quinone oxidation product of the monohydric phenol substrate, 4-hydroxyanisole, to mushroom tyr osinase. Column chromatography and SDS-PAGE separation showed labellin g of the enzyme when incubated with C-14 ring-labelled 4-hydroxyanisol e. It is proposed that covalent binding to the enzyme and other protei ns is through reaction of accessible nucleophilic groups, including th iols and amino groups, with the anisylquinone. This reductive addition enables the indirect generation of the catecholic substrate, which ac ts as an electron donor for the bicupric active site of met-tyrosinase and explains the lag kinetics of tyrosinase oxidation of non-cyclizin g substrates. The effects of diluting the enzyme or the addition of am ino acids on the lag period was consistent with a mechanism involving indirect generation of the dihydric phenol, which acts as the met-enzy me-recruiting substrate.