The activity of the type 3 copper enzyme tyrosinase toward 2-, 3-, and 4-fluorophenol was studied by kinetic methods and H-1 and F-19 NMR spectroscopy. Whereas 3- and 4-fluorophenol react with... Show moreThe activity of the type 3 copper enzyme tyrosinase toward 2-, 3-, and 4-fluorophenol was studied by kinetic methods and H-1 and F-19 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 fluoro-quinone, 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-1 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. Show less
The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and... Show moreThe double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman). Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed CyS42 assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112). A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands. The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell. Show less
