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Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine, N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period. N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation.Evidence that the indoliumolate product is formed by cyclization of the ortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s ؊1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue, N,N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived from N,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.Tyrosinase (EC 1.14.18.1) is an enzyme widely distributed in nature that catalyzes the oxidation of monohydric phenols (such as tyrosine). It exhibits unusual kinetic properties. Its natural substrate is considered to be tyrosine, yet it exhibits an induction period or a lag phase in the oxidation of this substrate (1). The lag phase is explained by an autocatalytic mechanism that depends on the elaboration of dihydroxyphenylalanine (DOPA) 1 in the initial phase of the reaction pathway of melanogenesis. There are two main mechanistic theories of tyrosinase autocatalysis: (a) allosteric activation and (b) the recruitment hypothesis, which depends on the two-electron reduction of the active site of the enzyme by the oxidation of dihydroxy substrates. There has been some controversy in the literature regarding the method of generation of DOPA and, therefore, of the explanation of the kinetics. According to on...

A pulse radiolytic investigation has been conducted to establish whether a redox reaction takes place between dopaquinone and 5,6-dihydroxyindole (DHI) and its 2-carboxylic acid (DHICA) and to measure the rate constants of the interactions. To obviate possible confounding reactions, such as nucleophilic addition, the method employed to generate dopaquinone used the dibromide radical anion acting on dopa to form the semiquinone which rapidly disproportionates to dopaquinone. In the presence of DHI the corresponding indole-5,6-quinone (and/or tautomers) was also formed directly but, by judicious selection of suitable relative concentrations of initial reactants, we were able to detect the formation of additional indolequinone from the redox exchange reaction of DHI with dopaquinone which exhibited a linear dependency on the concentration of DHI. Computer simulation of the experimental time profiles of the absorption changes showed that, under the conditions chosen, redox exchange does proceed but not quite to completion, a forward rate constant of 1.4 x 10(6)/M/s being obtained. This is in the same range as the rate constants previously established for reactions of dopaquinone with cyclodopa and cysteinyldopa. In similar experiments carried out with DHICA, the reaction more obviously does not go to completion and is much slower, k (forward) =1.6 x 10(5)/M/s. We conclude that, in the eumelanogenic pathway, DHI oxidation may take place by redox exchange with dopaquinone, although such a reaction is likely to be less efficient for DHICA.

Melanins are the principal surface pigments in vertebrates and, in humans, play a major role in photoprotection. Although the product (melanin) has a mainly protective function in the skin, the process of melanogenesis represents a potential cellular hazard and is confined to special membrane-limited organelles (melanosomes) in a set of specialized dendritic cells (melanocytes) which synthesize the pigment and transfer it to recipient cells. Malignant melanocytes tend to exhibit up-regulated melanogenesis and defective melanosomes. These features suggest ways in which anti-melanoma therapy may be specifically targeted. Two general chemotherapeutic modalities are considered: 1 The 'Achilles heel' approach in which the generation of reactive quinones capable of leaking into the cytosolic compartment and causing structural and functional derangement is encouraged by the use of analogue substrates. 2 The 'Trojan horse' approach, in which a cytotoxic agent is selectively released by a tyrosinase-dependent mechanism.