Tyrosinase hydroxylase

Copper is part of several essential enzymes including tyrosinase (melanin production), dopamine beta-hydroxylase (catecholamine production), copper-zinc superoxide dismutase (free radical detoxification), and cytochrome oxidase and ceruloplasmin (iron conversion) (Aaseth and Norseth 1986). All terrestrial animals contain copper as a constituent of cytochrome c oxidase, monophenol oxidase, plasma monoamine oxidase, and copper protein complexes (Schroeder et al. 1966). Excess copper causes a variety of toxic effects, including altered permeability of cellular membranes. The primary target for free cupric ions in the cellular membranes are thiol groups that reduce cupric (Cu+2) to cuprous (Cu+1) upon simultaneous oxidation to disulfides in the membrane. Cuprous ions are reoxidized to Cu+2 in the presence of molecular oxygen molecular oxygen is thereby converted to the toxic superoxide radical O2, which induces lipoperoxidation (Aaseth and Norseth 1986). 

Copper is a component of many enzymes including amine oxidase, lysyl oxidase, ferroxidase, cytochrome oxidase, dopamine P-hydroxylase, superoxide dismutase and tyrosinase. This latter enzyme is present in melanocytes and is important in formation of melanin controlling the colour of skin, hair and eyes. Deficiency of tyrosinase in skin leads to albinism. Cu " ion plays an important role in collagen formation. 

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). 

Tyrosinase is both an oxidase and a hydroxylase. Some other copper enzymes have only a hydroxylase function. One of the best understood of these is the peptidylglycine a-hydroxylating monoxygenase, which catalyzes the first step of the reaction of Eq. 10-11. The enzyme is a colorless two-copper protein but the copper atoms are 1.1 nm apart and do not form a binuclear center.570 Ascorbate is an essential cosubstrate, with two molecules being oxidized to the semidehydro-ascorbate radical as both coppers are reduced to Cu(I). A ternary complex of reduced enzyme, peptide, and 02 is formed and reacts to give the hydroxylated product.570 A related two-copper enzyme is dopamine (J-monooxygenase, which utilizes 02 and ascorbate to hydroxylate dopamine to noradrenaline (Chapter 25).571/572 These and other types of hydroxylases are compared in Chapter 18. 

Copper has an essential role in a number of enzymes, notably those involved in the catalysis of electron transfer and in the transport of dioxygen and the catalysis of its reactions. The latter topic is discussed in Section 62.1.12. Hemocyanin, the copper-containing dioxygen carrier, is considered in Section 62.1.12.3.8, while the important role of copper in oxidases is exemplified in cytochrome oxidase, the terminal member of the mitochondrial electron-transfer chain (62.1.12.4), the multicopper blue oxidases such as laccase, ascorbate oxidase and ceruloplasmin (62.1.12.6) and the non-blue oxidases (62.12.7). Copper is also involved in the Cu/Zn-superoxide dismutases (62.1.12.8.1) and a number of hydroxylases, such as tyrosinase (62.1.12.11.2) and dopamine-jS-hydroxylase (62.1.12.11.3). Tyrosinase and hemocyanin have similar binuclear copper centres. 

Several diverse metal centres are involved in the catalysis of monooxygenation or hydroxylation reactions. The most important of these is cytochrome P-450, a hemoprotein with a cysteine residue as an axial ligand. Tyrosinase involves a coupled binuclear copper site, while dopamine jS-hydroxylase is also a copper protein but probably involves four binuclear copper sites, which are different from the tyrosinase sites. Putidamonooxin involves an iron-sulfur protein and a non-heme iron. In all cases a peroxo complex appears to be the active species. 

The phenol-oxidizing enzyme tyrosinase has two types of activity (/) phenol o-hydroxylase (cresolase) activity, whereby a monophenol is converted into an o-diphenol via the incorporation of oxygen, and (2) cathecholase activity, whereby the diphenol is oxidized. The two reactions are illustrated in Figure 2-6, in the conversion of tyrosine (2.40) to L-DOPA (3,4-dihydroxyphenylalanine (2.41), dopaquinone (2.42), and indole-5,6-quinone carboxylate (2.43), which is further converted to the brown pigment... 

This enzyme exhibits no hydroxylase activity and is involved in the final synthesis of many naturally occurring /t-quinoncs. e.g. the naphthaquinone juglone in walnut (1.58) and the benzoquinone arbutin (hydroquinone-(3-D-glucopyranoside 2.46). Arbutin is a plant cryo-protectant that stabilizes membranes (Hincha et al., 1999). This compound has medicinal properties and has, for example, been used to treat urinary tract infections in humans. It is also used to lighten skin color, because it inhibits tyrosinase and hence the formation of melanin. The derivative deoxyarbutin (2.47 note the difference in the sugar molecule) was recently reported to be considerably more effective as a skin-lightening compound (Boissy et al., 2005). 

Oxygenation tyrosinase, dopamine /3-hydroxylase, phenylalanine hydroxylase, peptidylglycine a-amidating monooxygenase... 

In vivo tolerance to copper is quite high, however, deficiency and excess are serious problems. Infants are particularly vulnerable as they take time to assimilate the correct levels and it is known that trace copper from cooking utensils or water pipes can cause childhood cirrhosis. Copper deficiency leads to arterial weakness and heart enlargement. This is probably caused by a decrease in catecholamine neurotransmitters derived from the biosynthesis of adrenaline which requires the copper-containing enzymes phenylalanine hydroxylase, dopamine P-monooxygenase and tyrosinase. 

Enzyme deficiency diseases. A variety of metabolic diseases are caused by deficiencies or malfunctions of enzymes, due originally to gene mutation. Albinism, for example, may be caused by the absence of tyrosinase, an enzyme essential for the production of cellular pigments. The hereditary lack of phenylalanine hydroxylase results in the disease phenylketonuria (PKU) PKU is usually managed by dietary modifications, but intravenous... 

The clinical symptoms of classical Menkes disease can be traced back to developmentaUy important copper enzymes such as lysyl oxidase, tyrosinase (see Copper Hemocyanin/Tyrosinase Models), cytochrome c oxidase (see Cytochrome Oxidase), dopamine -hydroxylase, superoxide dismutase, and amine oxidase (see Superoxide Dismutase). Lysyl oxidase is needed for the cross-linking of connective tissue a deficiency in this enzyme causes weakened connective tissue and connective tissue disorder such as arterial ruptures as observed in these patients. Low levels of cytochrome c oxidase cause temperature instability and the absence of tyrosinase explains the hair depigmentation observed in affected individuals. ... 

A number of copper requiring enzymes are located at the cell surface or are exported into the extracellular milieu. Examples of such secretory Cu-enzymes include copper requiring ferroxidases that fimction in iron transport (e g. ceruloplasmin, CP), enzymes for neurotransmission (peptidyl amidating enzyme and dopamine hydroxylase), an extracellular superoxide dismutase (SOD) that fimctions in antioxidant defense and enzymes for formation of connective tissue (lysyl oxidase), and pigments (tyrosinase) (reviewed in ). En route to their designated location, each of these enzymes passes through a specialized compartment of the late Golgi where copper insertion takes place. 

Tyrosinase is a copper-containing oxidase (Coche-Guerente et al, 2001 Forzani et al, 2000), which possesses the two different activities illustrated in Figure 57.12. In the first step, referred to as the hydroxylase or cresolase activity, molecular oxygen is used to hydroxylate phenol to form catechol. In the second step, known as the catecholase activity, the enzyme oxidizes catechol to o-quinone, which is simultaneously oxidized by oxygen to its original form, with the production of water. The o-quinone is electro-chemically active and can be reduced back to catechol, as illustrated above in Eq. (57.17). 

Active sites containing two copper ions that are antiferromagneti-cally coupled in the oxidized state are often referred to as type 3 copper sites (129). It has recently become evident that these centers cannot all be considered alike and that in the blue copper oxidases the type 3 sites are in fact part of a tricopper cluster these will be considered in Section VA. The proteins containing dinuclear type 3 copper sites comprise hemocyanin and a number of oxygenase enzymes, of which the best known are tyrosinase and dopamine j8-hydroxylase. 

Catechol as an Activator of Tyrosinase. The phenolase activity of tyrosinase has been studied less completely than the catecholase activity, partly because of the lack of a satisfactory assay procedure. The phenolase reaction, however, is characterized by a lag time which can be abolished by adding dihydroxyphenylalanine (DOPA), the immediate product of the hydroxylation reaction 29S4, 102, 117), This phenomenon has been described by several investigators (29-34) and is illustrated in Figure 12, from Pomerantz and Warner (117), using the enzyme from Hamster melanoma. The same phenomenon has been analyzed by Duckworth and Coleman (102) for the mushroom enzyme. In the absence of DOPA, maximum velocity of the hydroxylase reaction is not reached for several minutes. Pomerantz and Warner (117) devised a convenient assay for the phenolase reaction by determining the radio-... 

Tyrosinase is both an oxidase and a hydroxylase. Some other copper enzymes have only a hydroxylase fxmction. One of fhe best understood of these is the peptidylg ycine a-hydroxylating monoxygenase,... 

Japanese publications report that vitamin E can be used elfectively in the treatment of facial pigmentation. It inhibits tyrosinase indirectly by inhibiting its hydroxylase activity. It also acts by limiting the oxidative phases required in the first stages of the transformation of tyrosine into indole derivatives. Vitamin E oxidizes easily, and more stable derivatives than pure a-tocopherol have to be used in cosmetic preparations. Tocopheryl acetate is one of the more stable derivatives of vitamin E. Tocopheryl ferulate is also frequently used. 

A. The correct response is dihydropteridine reductase. This enzyme reduces dihydrobiopterin to tetrahydrobiopterin the obligate electron donor for phenylalanine hydroxylase. Tyrosinase is the first enzyme on the pathway to melanin. Dopamine hydroxylase and tyrosine transaminase are enzymes on other tyrosine metabohc tracts. Homogentisic acid oxidase is an enzyme on the pathway of tyrosine to fumarate and acetoacetate. 

For the determination of phenols and amines enzymes with low selectivity, e.g. laccase, horseradish peroxidase, tyrosinase, and polyphenol oxidase, as well as specific enzymes, e.g. phenol hydroxylase and catechol-1,2-oxygenase, can be used in biosensors. 

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