Tyrosinase action

Some of the spectral effects of specific covalent modifications of proteins, i.e., iodination, oxidation, tyrosinase action, etc., will be discussed briefly. Heme proteins, flavoproteins, and other conjugated proteins will not be discussed except as regards studies involving their amino acid components. 

Other DFT-B3LYP calculations were performed by Siegbahn and coworkers [343], this time on a neutral model system L3CU. .. Cu L3 to probe the mechanism of tyrosinase action. The ligands L chosen to model histidines, were either ammonia or formimine. The authors focus on the choice of chemical model and its limitations, the location of the transition state for 0-0 activation... 

More often than not, it takes 3-4 months of daily applications of AHA creams alone to get a clinical result. AHAs are not renowned for their anti-tyrosinase action they limit melanin synthesis mainly through their antioxidant power and increase the turnover and renewal of cells laden with pigment. Using just AHA creams as the only form of treatment for melasma is disappointing. 

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

Ortho-hydroxylations by copper complexes mimicking tyrosinase action take place probably via intervention of p-peroxocopper(III) species. 

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. 

Dianisyltellurium oxide (DAT) is a mild and selective oxidant for quinone for-mation. Treatment of the A,A-di-n-propyldopamine (2) with DAT leads to the betaine (3), which is identical with the product of oxidation by the enzyme tyrosinase both of (2) and of the monohydric phenol A,A-dimethyltyramine. The implications and relevance to the mode of action of tyrosinases have been discussed. 

Tyrosinase is a monooxygenase which catalyzes the incorporation of one oxygen atom from dioxygen into phenols and further oxidizes the catechols formed to o-quinones (oxidase action). A comparison of spectral (EPR, electronic absorption, CD, and resonance Raman) properties of oxy-tyrosinase and its derivatives with those of oxy-Hc establishes a close similarity of the active site structures in these proteins (26-29). Thus, it seems likely that there is a close relationship between the binding of dioxygen and the ability to "activate" it for reaction and incoiporation into organic substrates. Other important copper monooxygenases which are however of lesser relevance to the model studies discussed below include dopamine p-hydroxylase (16,30) and a recently described copper-dependent phenylalanine hydroxylase (31). 

Mecfianism of Action The mechanism of action is not fully understood. Monobenzone maybe converted to hydroquinone, which inhibits the enzymatic oxidation of tyrosine to DOPA it may have a direct action on tyrosinase, or it may act as an antioxidant to prevent SH-group oxidation so that more SH groups are available to inhibit tyrosinase. Therapeutic Effect Depigmentation in extensive vitiligo. 

The mechanism of action of these compounds appears to involve inhibition of the enzyme tyrosinase, thus interfering with the biosynthesis of melanin. In addition, monobenzone may be toxic to melanocytes, resulting in permanent loss of these cells. Some percutaneous absorption of these compounds takes place, because monobenzone may cause hypopigmentation at sites distant from the area of application. Both hydroquinone and monobenzone may cause local irritation. Allergic sensitization to these compounds can occur. Prescription combinations of hydroquinone, fluocinolone... 

Before kinetic constants can be evaluated, it is critical to find the correct concentration of enzyme to use for the assays. If too little enzyme is used, the overall absorbance change for a reaction time period will be so small that it is difficult to detect differences due to substrate concentration changes or inhibitor action. On the other hand, too much enzyme will allow the reaction to proceed too rapidly, and the leveling off of the time course curve as shown in Figure E5.7 will occur very early because of the rapid disappearance of substrate. A rate that is intermediate between these two extremes is best. For the dopachrome assay, it is desirable to use the level of tyrosinase that gives a linear absorbance change at 475 nm for 2 minutes. 

Melanin granules are secreted by melanocytes in the hair papilla and distributed to keratin in the hair cortex and inner layers of the hair sheath during normal development. Melanogenesis is subject to hormonal control and has been the focus of intensive genetic studies. Two main forms of melanin exist in human skin—eumelanin and phaeomelanin, both of which are derived from tyrosine through the action of tyrosinase (a cupro-enzyme) and possibly other key enzymes (with nickel, chromium, iron, and manganese as cofactors). Tyrosine is converted to dihydroxyphenylalanine and, via a series of intermediate steps, to indole-5,6-quinone, which polymerizes to eumelanin. Phaeomelanins are produced by a similar mechanism but with the incorporation of sulfur (as cysteine) by a nonenzymatic step in the oxidation process. 

The great specificity of tyrosinase, the simplicity of monitoring its action spectrally, and in some cases, the discrimination shown among the several tyrosyl groups of a protein [it oxidizes only one of the six tyrosyl groups of ribonuclease (Yasuiiobu and Dandliker, 1957)] appear to recommend its serious consideration as a tool in protein structure studies. 

While many of these copper oxidases are essential to life as we know it on this planet, and some, like tyrosinase, are essential to our own health, their mechanism of action has remained relatively obscure, certainly in relation to our knowledge of some of the proteolytic enzymes. This paper has attempted to outline the considerable progress that has been made over the past several years in understanding these difficult enzymes. Much of the new information has been developed because of the presence of copper as a reporter group at the active site. 

Mechanistic aspects of the action of tyrosinase and the usual transduction schemes have been summarized on several occasions [166,170-173]. In short, this copper enzyme possesses two activities, mono- and di-phenolase. Due to the predominant presence of the mono phenolase inactive form (met-form), the enzyme is inherently inefficient for the catalysis of these monophenol derivatives. However, in the presence of a diphenol, the catalytic cycle is activated to produce quinones and the scheme results in an efficient biorecognition cascade. This activation is achieved more efficiently when combined with electrochemical detection through the reduction of the produced quinones [166], as illustrated in Fig. 10.5. Consequently, a change in the rate-hmiting step can be observed through kinetic to diffusion controlled sensors with a concomitant increase in stability and sensitivity, as depicted in Fig. 10.6. 

Abrol et al. (637), reporting on the darkening of whole wheat meal, due to the action of tyrosinase in bran, found added ascorbic acid (0.5-2.0 mg/kg) to retard the darkening effect. In another context, durum and other wheat granular flours (semolina) require retention of their natural yellow carotenoid content for the manufacture of pasta products which Dahle (638) and Walsh et al. (639) observed were stabilized by added L-ascorbic acid. 

As a general rule, it is worthwhile preparing the skin carefully with tyrosinase inhibitors if there is any risk of post-peel pigmentary changes or to optimize results when treating melasma. Retinoic acid and sometimes glycolic acid are used to make transepidermal penetration more even or to deepen the action of the acid solution. 

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