Tyrosinase action on monophenols: evidence for direct enzymatic release ofo-diphenol

Using gas chromatography-mass spectrometry, the direct enzymatic release of o-diphenol (4-tert-butylcatechol) during the action of tyrosinase on a mon ophenol (4-tert-butylphenol) has been demonstrated for the first time in th e literature. The findings confirm the previously proposed mechanism to exp lain the action of tyrosinase on monophenols (J.N. Rodriguez-Lopez, J. Tude la, R. Varon, F. Garcia-Carmona, F. Garcia-Canovas, J. Biol. Chem. 267 (199 2)). Oxytyrosinase, the oxidized form of the enzyme with a peroxide group, is the only form capable of catalysing the transformation of monophenols in to diphenols, giving rise to an enzyme-substrate complex in the process. Th e o-diphenol formed is then released from the enzyme-substrate complex or o xidized to the corresponding o-quinone. In order to detect the enzymatic re lease of o-diphenol, the non-enzymatic evolution of the o-quinone to genera te o-diphenol by weak nucleophilic attack reactions and subsequent oxidatio n-reduction was blocked by the nucleophilic attack of an excess of cysteine . Furthermore, the addition of catalytic quantities of an auxiliary o-diphe nol (e.g. catechol) considerably increases the accumulation of 4-tert-butyl catechol. The enzyme acting on 4-tert-butylphenol generates the enzyme-4-te rt-butylcatechol complex and 4-tert-butylcatechol is then released (with k( -2)) generating mettyrosinase. The auxiliary o-diphenol added (catechol) an d the 4-tert-butylcatechol generated by the enzyme then enter into competit ion. When [catechol] much greater than [4-tert-butylcatechol], the enzyme p referentially binds with the catechol to close the catalytic cycle, while 4 -tert-butylcatechol is accumulated in the medium. In conclusion, we demonst rate that the enzyme produces 4-tert-butylcatechol from 4-tert-butylphenol, the concentration of which increases considerably in the presence of an au xiliary o-diphenol such as catechol. (C) 2001 Elsevier Science B.V. All rig hts reserved.