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 tyrosina se and peroxidase catalyzed oxida tions of a series of catecholic comp ounds using the electron spin resonance (ESR) spin-stabilization appro ach and in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBT H). In the tyrosinase process, the nonenzymatic o-semiquinone formatio n by inverse disproportion mechanism was clearly confirmed. Mechanisms and kinetic studies of o-semiquinone and o-quinone formation by mushr oom 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-dihydro xyphenyl)propionic acid) together with an uncyclizable substrate (3,4- dihydroxybenzoic acid) were examined The reactive quinones were monito red by measuring the apparent initial rates of the o-quinone-MBTH addu cts. The transient behaviour of the o-semiquinone was studied by deter mining the Pseudo first-order formation constants (k values in the ran ge O.226-O.O35 s(-1)), the relative second-order decay kinetic constan ts (k = 3.3.10(2) M-1 s(-1) for dopamine o-semiquinone) and the maximu m concentrations of the o-semiquinone complexes formed in situ with Mg 2+ ions. The o-semiquinone formation constants are not directly correl ated with their maximum concentrations; in fact, the o-semiquinone max imum concentration of the uncyclizable substrate is comparable with th at derived from L-dopa. Furthermore, the secondary semiquinone formati on is slow and not competitive with the primary semiquinone generation . Then, in oar model the limiting factor for the o-semiquinone formati on, is not simply the substrate ability to cyclize, anti, therefore, t he potential toxicity of the secondary semiquinone is questionable. (C ) 1997 Elsevier Science lnc.