Unravelling the suicide inactivation of tyrosinase: A discrimination between mechanisms

Abstract

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.