Abstract
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.
Highlights
Tyrosinases (Tys)1 are monooxygenating enzymes that catalyze the ortho-hydroxylation of monophenols and the subsequent oxidation of diphenols to quinones [1]
The type 3 site of Ty can exist in three main redox forms: (a) Tydeoxy [Cu(I) Cu(I)], the reduced species which binds oxygen to give (b) Tyoxy [Cu(II)-O22Ϫ-Cu(II)], in which molecular oxygen is bound as peroxide in a -2:2 side-on bridging mode, and (c) Tymet [Cu(II)-Cu(II)], the resting form of the enzyme, where the Cu2ϩ ions are normally bridged by a small ligand [7, 8]
In this paper we report a mechanistic and kinetic study on the activity of Ty from Streptomyces antibioticus toward the three isomeric fluorophenols, an important class of xenobiotic substances
Summary
Tyrosinase; L-dopa, 3,4-dihydroxyphenylalanine; FP, fluorophenol; m-FP, 3-fluorophenol; p-FP, 4-fluorophenol; o-FP, 2-fluorophenol; HPLC, high pressure liquid chromatography; ESI-MS, electrospray ionization-mass spectrometry. In catechol oxidases, which perform the oxidation of o-diphenols to quinones [5] The reasons why these proteins perform different functions, their catalytic sites appear to be similar both in structure and oxygen binding ability, remains to be clarified [6]. The tyrosinases present in soil have been found recently to be involved in the formation of humus through random coupling of different aromatic compounds. These findings have attracted interest in the activity of Ty as a potential detoxifying agent for xenobiotic compounds with phenolic structure [20, 21]. The number of fluorinecontaining environmental contaminants has strongly increased during the past decade [22], and the polymerization processes mediated by Tys can contribute to soil clean up and water treatment
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