Kinetic properties of hexameric tyrosinase from the crustacean Palinurus elephas.
Tyrosinases catalyze hydroxylation of monophenols to o-diphenols and their subsequent oxidation to o-quinones, whereas catecholoxidases catalyze only the latter reaction. Both enzymes occur in all organisms and are Type 3 copper proteins that perform the first steps of melanin formation. In arthropods, they play an essential role in the sclerotization of the exoskeleton. Very few phenoloxidases are characterized structurally or kinetically and the existence of an actual tyrosinase activity has not been demonstrated in most cases. Here we present for the first time a complete kinetic characterization of a tyrosinase from a crustacean (Palinurus elephas) including the influence of inhibitors. In contrast to most tyrosinases which are monomeric or dimeric, this tyrosinase occurs as a hexamer. However, the data did not indicate cooperativity in steady-state kinetics for the two substrates used, the monophenol tyramine and the diphenol dopamine. Mimosine as well as phenylthiourea (PTU) inhibited both monophenolhydroxylase and diphenoloxidase activity. Inhibition by mimosine was competitive, whereas PTU was a noncompetitive inhibitor. Furthermore, for the diphenolase activity substrate inhibition was observed, which was apparently abolished by adding PTU. These observations lead to the hypothesis that a secondary, allosteric binding site exists, which binds dopamine and PTU and reduces the catalytic activity.
Brack
Antje
A
Institut für Molekulare Biophysik, Johannes-Gutenberg-Universität, Mainz, Germany. brack@uni-mainz.de
Hellmann
Nadja
N
Decker
Heinz
H
D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D004298:Dopamine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D007700:Kinetics; D008898:Mimosine / Q000737:chemistry; D015394:Molecular Structure; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D033521:Palinuridae / Q000201:enzymology*; D010670:Phenylthiourea / Q000737:chemistry; D014439:Tyramine / Q000737:chemistry
D004791:Enzyme Inhibitors; D008898:Mimosine; D010670:Phenylthiourea; D014442:Monophenol Monooxygenase; D004298:Dopamine; D014439:Tyramine