Unravelling the suicide inactivation of tyrosinase: A discrimination between mechanisms

The suicide inactivation kinetics of tyrosinase acting on 3-isopropyl-6-methylcatechol, 3-tert-butyl-6-methylcatechol and 3,6-difluorocatechol was studied. All three substrates act as suicide substrates despite the fact that their 3 and 6 positions are occupied, confirming the mechanism proposed in Biochem. J. (2008) 416, 431–440. Although the most active substrate for the suicide inactivation was 3,6-difluorocatechol, its efficiency was much lower than that of the catechol used as reference. Its r value, the number of turnovers made by one mol of enzyme before its inactivation, is the highest described in the bibliography, highlighting the great difference between the catalytic and inactivation constants. A study of the effect of the pH on the enzymatic activity of tyrosinase showed that both 3-isopropyl-6-methylcatechol and 3-tert-butyl-6-methylcatechol behave as typical substrates of tyrosinase, while 3,6-difluorocatechol behaves differently. The remarkable behavior of 3,6-difluorocatechol when reacts with tyrosinase may be due to the fact that its two hydroxyl groups have very low pK values as a result of the strong electron-withdrawing effect of the fluorine atoms in the ortho positions, so that, at pH 7.0, the substrate would be mainly negatively charged. The apparent Michaelis constant shows a minimum value at pH 6.0, but increases at both high and low pH. However, the values of the catalytic constant and maximum apparent inactivation constant do not vary with the pH, so that the r remains practically constant. Under anaerobic conditions, 3,6-difluorocatechol acts as an irreversible inhibitor of the deoxy- and met-tyrosinase forms.

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► The compounds derived from catechol, 3,6-disubstituted, behave as suicide substrates of tyrosinase under aerobic conditions.
► The KmS of 3,6-difluorocatechol vs. pH follows a parabolic behavior.
► The action mechanism of 3,6-difluorocatechol points to the existence of two critical apparent pKs in the enzyme.
► The r-value of 3,6-difluorocatechol is the highest described in the literature.
► Under anaerobic conditions, 3,6-difluorocatechol irreversibly inactivates the enzyme.