Cresolases

Perez-Gilabert M and Garcia-Carmona F. 2000. Characterization of catecholase and cresolase activities of eggplant polyphenol oxidase. J Agric Food Chem 48(3) 695-700. 

Polyphenol oxidase occurs within certain mammalian tissues as well as both lower (46,47) and higher (48-55) plants. In mammalian systems, the enzyme as tyrosinase (56) plays a significant role in melanin synthesis. The PPO complex of higher plants consists of a cresolase, a cate-cholase and a laccase. These copper metalloproteins catalyze the one and two electron oxidations of phenols to quinones at the expense of 02. Polyphenol oxidase also occurs in certain fungi where it is involved in the metabolism of certain tree-synthesized phenolic compounds that have been implicated in disease resistance, wound healing, and anti-nutrative modification of plant proteins to discourage herbivory (53,55). This protocol presents the Triton X-114-mediated solubilization of Vida faba chloroplast polyphenol oxidase as performed by Hutcheson and Buchanan (57). 

This copper-dependent enzyme [EC 1.14.18.1] (also known as tyrosinase, phenolase, monophenol oxidase, and cresolase) catalyzes the reaction of L-tyrosine with L-dopa and dioxygen to produce L-dopa, dopaquinone, and water. This classification actually represents a set of copper proteins that also catalyze the reaction of catechol oxidase [EC 1.10.3.1] if only 1,2-benzenediols are available as substrates. 

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... 

Tyrosinase obtained relatively pure was tested for its effect in rats, dogs (53), and man (50), and was found to exert a specific depression of blood pressure only in hypertensive animals. Tyrosinase contains catecholase and cresolase, and has the property of... 

The phenol oxidases probably play no important role in the elimination of phenolic pressor amines, in spite of the importance that has been attached to the oxidation of the catechol nucleus in the past. The names phenolase and cresolase, polyphenol oxidase, and catechol oxidase serve to identify the enzyme with its mono- or diphenolic substrate, but they usually occur together and are difficultly separated. The enzymes have been purified and their characteristics have been described (56, 104, 106, 156). Beyer (21), Alles (5), and Randall and Hitchings (129) have described the relationship of structure of the phenolic pressor amines to the rate of oxidation of their nucleus in the presence of these enzymes. 

Crescenza cheeses 761 Cresol 522 Cresolase 371 Crosslinking 808 Crown ethers 57, 67 Crystallography 804 Cucumber (Cucumis sativus) 370 Current-voltage (I/V) 91 Cyanide 107, 109 Cyanobacterial cells el51 toxins 331... 

The substrate analog and spectroscopic studies led Solomon et al. to suggest a mechanism for the Tyr cresolase activity (Figure 2) [22], Here, a phenol substrate could bind initially to oxy-Tyr in an axial fashion, a possibility confirmed in model studies [28,29], In this ternary Cu2/02/substrate complex, rearrangement through a trigonal bipyramidal intermediate could be accompanied by ortho-hy-droxylation, followed by loss of water and coordination of the diphenol product. Such a catecholate dicopper(II) complex is known in model systems [30], Intramolecular electron transfer would result in release of product o-quinone and the dicopper(I) produced could react with 02 again to produce oxy-Tyr. 

To distinguish this type of activity from the one mentioned earlier, it is described as cresolase activity, whereas the other is referred to as catecholase activity. For both types of activity, the involvement of copper is essential. Copper has been found as a component of all polyphenolases. The activity of cresolase involves three steps, which can be represented by the following overall equation (Mason 1956) ... 

Tyrosinase catalyzes two reactions, the hydroxylation of phenolic compounds in ortho-position (cresolase activity) and subsequently the oxidation of the diphenolic products (cat-echolase activity).Tyrosinase as well as another enzyme that catalyzes only the oxidation reaction, catecholoxidase (EC 1.10.3.1), belongs to the group of phenoloxidases. The monooxygenase nature of Ty was established by Mason and coworkers in a pioneering study using 0-labeled oxygen. The two-electron donor required in the hydroxylation reaction is the o-diphenol, which is generated internally from the monophenol substrate. 

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). 

The dominant feature of tyrosinase is that it has both cresolase and catecholase activity. Laccase has a very clear catecholase activity, but its cresolase activity is not so clear. Mason, Fowlks, and Peterson (109) used 02 to label 3,4-dimethylphenol during the tyrosinase-catalyzed oxidation of this compound and showed that the source of the oxygen introduced into the phenol in the phenolase reaction was molecular oxygen according to Reaction 1. 

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is a bifunctional, copper-containing enzyme widely distributed on the phylogenetic tree. This enzyme uses molecular oxygen to catalyze the oxidation of monophenols to their corresponding o-diphenols (cresolase activity) as well as their subsequent oxidation to o-quinones (catecholase activity). The o-quinones thus generated polymerize to form melanin, through a series of subsequent enzymatic and nonenzymatic reactions [1-3]. 

The biosynthetic pathway for melanin formation, operating in insects, animals, and plants, has largely been elucidated by Raper [15], Mason [16], and Lerner et al. [17]. The first two steps in the pathway are the hydroxylation of monophenol to o-diphenol (monophenolase or cresolase activity) and the oxidation of diphenol to o-quinones (diphenolase or catecholase activity), both using molecular oxygen followed by a series of nonenzymatic steps resulting in the formation of melanin [15,18,19]. The whole pathway for melanin biosynthesis is shown in Scheme 1. 

By using lineweaver-Burk plots the authors found that four xanthates exhibited different patterns of mixed, competitive, or uncompetitive inhibition. For the cresolase activity, 1 and 2 demonstrated uncompetitive inhibition but 3 and 4 exhibited competitive inhibition [43]. For the catecholase activity, 1 and 2 showed mixed inhibition but 3 and 4 showed competitive inhibition against tyrosinase [43]. The xanthates (compoimds 1, 2, 3 and 4) have been classified as potent inhibitors against tyrosinase due to their Ki values of 13.8,... 

Catechol oxidase oxidizes otty o-diphenols to the corresponding quinones but lacks monooxygenase or cresolase activity. Hemocyanin acts as an oxygen carrier in arthropods and mollusks. 

The catalytic mechanism of tyrosinase was first studied in detail by Solomon et al Solomon proposed a mechanism for both the cresolase and catecholase activities of tyrosinase (Figure 25). This mechanism suggests the oxy state to be the starting point of cresolase activity (inner circle). This state is present in the resting form of tyrosinase in a proportion of about 15% (85% met state). A monophenol substrate binds to the oxy state and is monooxygenated to o-diphenol. This diphenol subsequently binds to the copper center of met tyrosinase in a... 

Figure 25 Mechanism of cresolase and catecholase activity of tyrosinase and catechoi oxidase deveioped on the basis of an initiai proposai by Soiomon and coworkers and inciuding more recent resuits.Reproduced from C. Gerdemann C. Eicken B. Krebs, Acc. Chem. Res. 2002, 35, 183-191, with permission from American Chemicai Society.

The distinct difference between catechol oxidase and tyrosinase has not yet been explained. A lag phase in the monophenolase activity of tyrosinase has been found and studied and is proposed to be a result of temporary inhibition of the met state of tyrosinase by excess of the monophenol substrate (Figure 25). Monophenolase activity increases when the diphenol product displaces the monophenol from met tyrosinase and allows the continuation of the catalytic cycle. Catechol oxidase in its isolated form is present exclusively in the met state and is also inhibited by phenol. It was therefore suggested that lack of the oxy state is the reason catechol oxidase lacks cresolase activity. As oxy catechol oxidase also shows no monooxygenase activity, this explanation does not seem entirely satisfying. Another possible reason is that access to Cu, which has been proposed to be necessary for the oxygenation of monophenols, is blocked in the crystal structure of catechol oxidase from... 

These two processes are also referred as cresolase activity or monophenolase activity and diphenolase activity, respectively. Such reactions represent the initial steps of vertebrate pigmentation (melanin biosynthesis) and the browning of fruits and vegetables.Catechol oxidases (EC 1.10.3.1) are ubiquitous plant enzymes, which also catalyze the oxidation of a broad... 

Polyphenol oxidases (e.g., tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, catecholase) Chitinolytic enzymes... 

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