Tyrosinase-catecholic substrates in vitro model: Kinetic studies on the o-quinone/o-semiquinone radical formation

The mechanism of o-semiquinone production was examined in the tyrosinase and peroxidase catalyzed oxida tions of a series of catecholic compounds using the electron spin resonance (ESR) spin-stabilization approach and in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). In the tyrosinase process, the nonenzymatic o-semiquinone formation by inverse disproportion mechanism was clearly confirmed. Mechanisms and kinetic studies of o-semiquinone and o-quinone formation by mushroom tyrosinase were carried out by ESR spin stabilization and optical spectroscopy. Two different types of cyclizable catecholic substrates (L-dopa and dopamine, 3,4-dihydroxyphenylacetic acid and 3(3,4-dihydroxyphenyl)propionic acid) together with an uncyclizable substrate (3,4-dihydroxybenzoic acid) were examined The reactive quinones were monitored by measuring the apparent initial rates of the o-quinone-MBTH adducts. The transient behaviour of the o-semiquinone was studied by determining the Pseudo first-order formation constants (k values in the range O.226-O.O35 s(-1)), the relative second-order decay kinetic constants (k = 3.3.10(2) M-1 s(-1) for dopamine o-semiquinone) and the maximum concentrations of the o-semiquinone complexes formed in situ with Mg2+ ions. The o-semiquinone formation constants are not directly correlated with their maximum concentrations; in fact, the o-semiquinone maximum concentration of the uncyclizable substrate is comparable with that derived from L-dopa. Furthermore, the secondary semiquinone formation is slow and not competitive with the primary semiquinone generation. Then, in oar model the limiting factor for the o-semiquinone formation, is not simply the substrate ability to cyclize, anti, therefore, the potential toxicity of the secondary semiquinone is questionable. (C) 1997 Elsevier Science lnc.