The activity of the type 3 copper enzyme tyrosinase toward 2-, 3-, and 4-fluorophenol was studied by kinetic methods and 1 H and 19 F NMR spectroscopy. Whereas 3-and 4-fluorophenol react with tyrosinase to give products that undergo a rapid polymerization process, 2-fluorophenol is not reactive and actually acts as a competitive inhibitor in the enzymatic oxidation of 3,4-dihydroxyphenylalanine (L-dopa). The tyrosinase-mediated polymerization of 3-and 4-fluorophenols has been studied in detail. It proceeds through a phenolic coupling pathway in which the common reactive fluoroquinone, produced stereospecifically by tyrosinase, eliminates an inorganic fluorine ion. The enzymatic reaction studied as a function of substrate concentration shows a prominent lag that is completely depleted in the presence of L-dopa. The kinetic parameters of the reactions can be correlated to the electronic and steric effects of the fluorine substituent position. Whereas the fluorine electron withdrawing effect appears to control the binding of the substrates (K m for 3-and 4-fluorophenols and K I for 2-fluorophenol), the k cat parameters do not follow the expected trend, indicating that in the transition state some additional steric effect rules the reactivity.
Tyrosinases (Tys)1 are monooxygenating enzymes that catalyze the ortho-hydroxylation of monophenols and the subsequent oxidation of diphenols to quinones (1). The reaction is widespread in nature, from bacteria to fungi, plants, and mammals. In mammals, when L-tyrosine acts as the substrate, the formed quinones are reactive precursors in the synthesis of melanin pigments. In fruits, vegetables, and mushrooms, Ty is a fundamental enzyme in the browning process that occurs during product storage and upon bruising. Ty contains a dinuclear type 3 copper center, in which two copper ions are closely spaced and coordinated each by three histidines through the N-⑀ nitrogen atoms (1). This type of site has been found and structurally characterized also in hemocyanins, which act as oxygen carriers in arthropods and mollusks (2-4), and in catechol oxidases, which perform the oxidation of o-diphenols to quinones (5). The reasons why these proteins perform different functions, although their catalytic sites appear to be similar both in structure and oxygen binding ability, remains to be clarified (6). During activity, the type 3 site of Ty can exist in three main redox forms: ions are normally bridged by a small ligand (7,8). The cloning technique, developed recently, and more detailed kinetic studies (6 -12) have increased the current understanding of Ty mechanism and structure. Additional studies (8, 13-16) on inhibitors also provided structural information. The importance of the latter studies is related to the considerable interest of Tys from the medical, agricultural, and industrial point of view. For instance, Ty inhibitors are of great concern in the cosmetic industry (17) and in food technology as anti-browning agents (18,19). Moreover, the tyrosinases present in soil have been fo...