Tyrosinase

The catalytic o-hydroxylation of phenols has biological relevance since the working catalyst systems may serve as models for tyrosinase, a copper-containing monooxygenase that effects oxygen insertion into the o-position of tyrosine (46) to produce dopa (47), which is then converted to dopamine (48) by dopa decarboxylase. The hydroxylation of dopamine leads to noradrenaline (49), which is an important neurotransmitter [138-141]. 

Tyrosinases from various sources also catalyze the hydroxylation of phenols to catechols and the oxidation of catechols to o-quinones [139]. 

The o-hydroxylation of phenols to produce o-benzoquinones is related to tyrosinase action [109,110], but is also of commercial interest. 

The following are examples of systems in which o-hydroxylation of phenol derivatives has been observed with various degrees of regioselectivity. 

Hydroquinone production is feasible via the copper-catalyzed oxidation of phenol with dioxygen to p-benzoquinone, with subsequent reduction [105,106], which can be effected using the same CuCl/acetonitrile catalyst under hydrogen. 

Li et al. (1990) developed an assay to measure the diphenol oxidase activity of tyrosine by following the conversion of 3,4-dihydroxymandelic acid (DHMA) to 3,4-dihydroxybenzaldehyde (DHBZ). Tyrosinase is involved in the formation of melanotic pigments in a wide variety of plants and animals. 

Separation of 3,4-dihydroxymandelic acid and 3,4-dihydroxybenzaldehyde occurred at 40°C on an Altex Ultrasphere ODS column (4.6 mm x 250 mm, 5 fim). The mobile phase at a flow rate of 1.0 mL/min consisted of 0.4 citrate buffer (pH 3.2) containing 0.5 mM Na2EDTA and 5% (v/v) acetonitrile. An amperometric detector fitted with a glassy carbon electrode was used. The electrode was maintained at +650 mV. 

The standard reaction mixture contained 0.3 fig of mushroom tyrosinase in 300 fiL of 0.05 M Mops buffer (pH 6.5) and 3.6 /tmol of 3,4-dihydroxybenzaldehyde dissolved in 300 fiL of the same buffer. The reaction mixture was incubated at 30°C, and aliquots were withdrawn at various times up to 10 minutes, with each added to an equal volume of ice-cold 0.2 M perchloric acid. After centrifugation, 2 to 5 fiL was analyzed by HPLC. For single time-point determinations, the reaction volume was reduced to 50 fiL. Product formation was linear with time to 10 minutes and with the amount of protein added. 

The assay was used to measure the activity of a commercial preparation of mushroom tyrosinase, and the activity in cell-free hemolymph from mosquitoes. 

The synthetase that catalyzes the synthesis of the named peptide from L-a-aminoadipic acid, L-cysteine, and L-valine uses ATP as the energy source. The enzyme activity is found in the actinomycete Streptomyces clavuligerus. 

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L-tyrosine phenol + pyruvic acid + NH P-tyrosinase Erwinia herbicola ... 

Genetic disruption of dopamine synthesis in mice lacking TH shows that dopamine is not essential for development. However, dopamine deficient mice do not survive long after weaning unless treated with l-DOPA. These mice display severe aphagia and adipsia and loss of motor function. While these mice have a major reduction in dopamine levels some residual dopamine can be detected that is generated through the action of tyrosinases. 

The early works by Muzzarelh et al. [179] showed that tyrosinase converts a wide range of phenohc substrates into electrophihc o-quinones [180]. Tyrosinase was used to convert phenols into reactive o-quinones which then underwent chemical reactions leading to grafting onto chitosan. A review article showed that in general the tyrosinase-catalyzed chitosan modifications resulted in dramatic changes in functional properties [181]. 

An interesting combination of enzymatic with non-enzymatic transformation in a one-pot three-step multiple sequence was reported by Waldmann and coworkers [82]. Phenols 125 in the presence of oxygen and enzyme tyrosinase are hydroxylated to catechols 126 which are then oxidized in situ to ortho quinones 127. These intermediates subsequently undergo a Diels-Alder reaction with inverse electron demand by reaction with different dienophiles (Table 4.19) to give endo bicyclic 1,2-diketones 128 and 129 in good yields. 

Table 4.19 Multiple hydroxylation-oxidation Diels-Alder reactions initiated by tyrosinase...

Among these factors, sun exposure remains the most important, although all of them significantly increase the activity of tyrosinase in producing melanin. 

Azelaic acid is a non-phenolic derivative (1,7-hep tanedicarboxylic acid) used at concentration of 10-20% twice a day to treat melasma with minimal side effects (allergic reactions). It acts to disturb the tyrosinase synthesis and can be used as a bleaching agent in patients sensitive to hydroquinone. Better results are obtained if a glycolic acid cream is applied sequentially to azelaic acid treatment. 

Kojic acid is a fungal metabolite (5-hydroxy-4 pyran 4-1-2 methyl) known to inhibit tyrosinase and used to treat melasma at concentration of 2-4% twice a day. The stability is one of its advantages if compared with hydroquinone. Unfortunately, it is considered to have a high sensitizing potential. 

Azelaic acid is a naturally occurring dicarboxyl-ic acid (1,7-heptanedicarboxylic acid) that has demonstrated beneficial therapeutic effects in the treatment of acne and several disorders of hyperpigmentation [48]. There are minimal effects on normally pigmented human skin, freckles, senile lentigines, and nevi. The cytotoxic and antiproliferative effects of azelaic acid may be mediated via inhibition of mitochondrial ox-idoreductase activity and DNA synthesis. Disturbance of tyrosinase synthesis by azelaic acid may also influence its therapeutic effects. Azelaic acid can be used as a hypopigmenting agent in patients sensitive to hydroquinone. 

Kojic acid (5-hydroxy-4 pyran 4-1-2 methyl) is a fungal derivative which inactivates tyrosinase via chelation of copper. Concentrations range from 2 to 4%. It can be used for monotherapy or in combination with retinoids or other cosme-ceutical products such as glycolic acid. Compared with hydroquinone, these kojic acid formulations usually show less efficacy. However, they may be effective in patients who do not... 

The oxidation of phenol in alcoholic media by a morpholine complex of Cu(II) (as a model for tyrosinase) to give 4,5-dimorpholino-orr/jo-benzoquinone in 64 %... 

Gandia-Herrero, R, Escribano, J., and Garci a-Carmona, R, Betaxanthins as substrates for tyrosinase an approach to the role of tyrosinase in the biosynthetic pathway of betalains. Plant Physiol, 138, 421, 2005. 

Steiner, U., Schhemann, W., and Strack, D., Assay for tyrosine hydroxylation activity of tyrosinase from betalain-forming plants and cell cultures, Anal. Biochem., 238, 72, 1996. 

Melanin biosynthesis in animals is a complex process starting with the L-tyrosine amino acid. In the first step, L-tyrosine is converted first into DOPA and then into dopaquinone, a process catalyzed by tyrosinase. In the biosynthesis of eumelanins, dopaquinone undergoes a cyclization to form dopachrome and subsequently a tau-tomerization into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). DHICA is further oxidized to indole-5,6-quinone2-carboxylic acid, the precnrsor of DHICA eumelanins. Tyrosinase-related proteins TRP-2 and TRP-1, respectively, are responsible for the last two steps, and they are under the control of the tyrosinase promoter. 

Murisier, F. and Beerman, F., Genetic of pigment cells lessons from the tyrosinase gene family, Histol. HistopathoL, 21, 567, 2006. 

Gandia-Herrero, P., Escribano, J., and Garcla-Carmona, P., Characterization of the activity of tyrosinase on betaxanthins derived from (iJ)-amino acids, J. Agric. Food Chem., 53, 9207, 2005. 

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