Tyrosinase activity

Fig. 5 Molecular structures of tyrosinase-active diterpene (8-10) and napelline (11, 12) type alkaloids [49]...

Copper(II) complexes with phenoxo ligands have attracted great interest, in order to develop basic coordination chemistry for their possible use as models for tyrosinase activity (dimeric complexes) and fungal enzyme galactose oxidase (GO) (monomeric complexes). The latter enzyme catalyzes the two-electron oxidation of primary alcohols with dioxygen to yield aldehyde and... 

Ross, P. K., and E. I. Solomon. 1991. An Electronic Structural Comparison of Cooper-Peroxide Complexes of Relevance to Hemocyanin and Tyrosinase Active Sites. J. Am. Chem. Soc. 113, 3246. 

The reaction of binuclear copper complexes with oxygen as models for tyrosinase activity was also markedly accelerated by applying pressure (106408 ). Tyrosinase is a dinuclear copper protein which catalyses the hydroxylation of phenols. This reaction was first successfully modeled by Karlin and co-workers (109), who found that an intramolecular hydroxylation occurred when the binuclear Cu(I) complex of XYL-H was treated with oxygen (Scheme 5). 

Fukuzumi and co-workers described spectroscopic evidence for a ix-rf- ] -peroxo-(Cu )2 species stabilized with a fcidentate nitrogen ligand, but no (catalytic) oxidation behavior towards catechol was noted (a related trinu-clear copper species converted 2,4-di-ferf-butylphenol stoichiometrically towards the biphenol derivative) [224], Stack et al. have described a similar ] -peroxo-(Cu )2 species (28, vide supra) that could be considered a structural and functional model for tyrosinase-activity, as it efficiently reacted with catechol, benzyl alcohol and benzylamine to yield quinone (95%), benzaldehyde (80%) and benzonitrile (70%) [172,173]. This dinuclear per-0X0 species is generated by association of two monomeric copper centers, in contrast to the systems based on dinucleating Ugand scaffolds described above. 

Albinism refers to a group of conditions in which a defect in tyrosine metabolism results in a deficiency in the production of melanin. These defects result in the partial or full absence of pigment from the skin, hair, and eyes. Albinism appears in different forms, and it may be inherited by one of several modes autosomal recessive, autosomal dominant, or X-linked. Complete albinism (also called tyrosinase-negative oculocutaneous albinism) results from a defi ciency of tyrosinase activity, causing a total absence of pigment from the hair, eyes, and skin (Figure 20.20). It is the most severe form of the condition. Affected people may appear to have white hair, skin, and iris color, and they may have vision defects. They also have photophobia (sunlight is painful to their eyes), they sun burn easily, and do not tan. 

Several kinetic characteristics of mushroom tyrosinase will be examined in this experiment. A spectrophotometric assay of tyrosinase activity will be introduced and applied to the evaluation of substrate specificity, Ku of the natural substrate, 3,4-dihydroxyphenylalanine (L-dopa), and inhibition characteristics. 

Many assays for tyrosinase activity have been developed. Procedures in the literature include use of the oxygen electrode, oxidation of tyrosine followed at 280 nm, and oxidation of dopa followed at 475 nm. The most convenient assay involves following the tyrosinase-catalyzed oxidation of dopa by monitoring the initial rate of formation of dopachrome at 475 nm (Figure E5.8). 

Now that the appropriate enzyme level has been determined, the kinetic constants may be evaluated. The Ku for L-dopa can be obtained by setting up the same assay as in part B, except that the factor to vary will be the concentration of L-dopa. The concentration of L-dopa in part B was sufficient to saturate all the tyrosinase active sites, so the rate depended only on the enzyme concentration. In part C, L-dopa levels will be varied over a range that is nonsaturating. 

Whether an inhibitor acts in a competitive or noncompetitive manner is deduced from a Lineweaver-Burk or direct linear plot using varying concentrations of inhibitor and substrate. In separate assays, two substances will be added to the dopa-tyrosinase reaction mixture, and the effect on enzyme activity will be quantified. The structures of the potential inhibitors, cinnamic acid and thiourea, are shown in Figure E5.9. The inhibition assays must be done immediately following the KM studies. To measure inhibition, reaction rates both with and without inhibitor must be used and the tyrosinase activity must not be significantly different. If it is necessary to do the inhibition studies later, the Ku assay for L-dopa must be repeated with freshly prepared tyrosinase solution. 

The mechanism of iris pigmentation due to latanoprost is unknown. In an in vitro experiment using uveal melanocytes, the addition of latanoprost increased melanin content, melanin production, and tyrosinase activity (18). Alpha-methyl-para-tyrosine, an inhibitor of tyrosinase (the enzyme that transforms tyrosine to levodopa), completely prevented the latanoprost-induced stimulation of melanogenesis. 

Humic acids have been shown to slightly inhibit tyrosinase activity by complexing the enzyme (Ruggiero and Radogna, 1988). Allison (2006b) also demonstrated that the addition of humic acid to a soil significantly decreased the polyphenoloxidase activity of the soil. 

Zamora, R., and Hidalgo, F. J. (2005). Coordinate contribution of lipid oxidation and Maillard reaction to the nonenzymatic food browning. Crit. Rev. Food Sci. Nutr. 45,49-59. Zavarzina, A. G., and Zavarzin, A. A. (2006). Laccase and tyrosinase activities in lichens. 

Mimicking and understanding tyrosinase activity (o-hydroxylation of phenols) have been of longtime interest because this was one of the earliest copper monooxygenases described and the significance of elucidating dioxygen activation mechanism(s) has widespread implications and potential applications. 

The expression product of pG3TY was also isolated by PAGE at pH 9.4 gel [139], The activity band of tyrosinase was stained with dihydroxy-L-phenylalanine (l-DOPA) after acidifying at pH 3. Tyrosinase activity of the pG3YT product was determined to be 0.018 nkat/kg protein. Without acidifying treatment at pH 3.0, no tyrosinase activity was observed. It can be concluded that the expression product of the melO gene is the protyrosinase of A. oryzae. 

Tyrosinase inhibitors prevent browning in foodbecause they inhibit the oxidation caused by the enzyme tyrosinase. Cuminaldehyde is identified as a potent mushroom tyrosinase monophenol monooxygenase inhibitor from cumin seeds, ft inhibits the oxidation of L-3,4-dihydroxyphenylalanine (l-DOPA) by mushroom tyrosinase with an ID50 of 7.7g/ml (0.05 mM). Its oxidized analogue, cumic acid (p-isopropylbenzoic acid), also inhibits this oxidation with an 1D50 of 43g/ml (0.26mM). These two inhibitors affect mushroom tyrosinase activity in different ways (Kubo and Kinst-Hori, 1998). 

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