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Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide substrates of tyrosinase Proteins and proteomics

Muñoz-Muñoz, Jose Luis, Garcia-Molina, Francisco, Berna, Jose, Garcia-Ruiz, Pedro Antonio, Varon, Ramon, Tudela, Jose, Rodriguez-Lopez, Jose N., Garcia-Canovas, Francisco
Biochimica et biophysica acta 2012 v.1824 no.4 pp. 647-655
enzyme inactivation, nitrogen, oxygen
We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamines<o-aminophenols<o-diphenols, which agrees with the view that the transfer of the proton to the peroxide group of the oxy-tyrosinase form is the slowest step in the catalytic cycle. As regards the apparent inactivation constant, it remains within the same order of magnitude, although slightly lower in the case of the aromatic o-diamines and o-aminophenols than o-diphenols: o-diamines<o-aminophenols<o-diphenols. The efficiency of the second nucleophilic attack of substrate on CuA seems to be the determining factor in the bifurcation of the inactivation and catalytic pathways. This attack is more efficient in o-diamines (where it attacks a nitrogen atom) than in o-aminophenols and o-diphenols (where it attacks an oxygen atom), favouring the catalytic pathway and slowing down the inactivation pathway. The inactivation step is the slowest of the whole process. The values of r, the number of turnovers that 1mol of enzyme carries out before being inactivated, follows the order aromatic o-diamines<o-aminophenols<o-diphenols. As regards the Michaelis constants, that of the o-diamines is slightly lower than that of the o-diphenols, while that of the o-aminophenols is slightly greater than that observed for the o-diphenols. As a consequence of the above, the inactivation efficiency, λₘₐₓ/Kₘ S, follows this order: o-diphenols>o-aminophenols>aromatic o-diamines.