Activity cresolase

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. 

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... 

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... 

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... 

Tyrosinase is a copper-containing glycoprotein that carries a coupled binuclear copper active site capable of catalyzing two distinct reactions (i) hydroxylation of tyrosine to dihydroxyphenylalanin, i.e. dopa (cresolase activity), and (ii) subsequent two-electron oxidation to dopaquinone (catecholase activity) (227). Both reactions require oxygen and the enzyme, i.e. tyrosinase, in reduced cuprous form. Because of these two catalytic functions, tyrosinase is in modern terminology referred to as a mixed function oxidase (757). A mechanism (Fig. 3) for hydroxylation... 

The enzyme tyrosinase catalyzes the oxidation of catechols to o-quinones (catecholase activity) and also the hydroxylation of phenols to o-diphenols (cresolase activity). This multisubunit enzyme contains several copper(I) sites in the active form. 

Tyrosinase. Tyrosinase is a very well-studied multicopper oxygenase, which contains a magnetically coupled dinuclear copper center. This enzyme catalyzes the ortho-hydroxylation of phenols to catechols (phenolase activity, also called cresolase activity), as well as the 2-electron oxidation of catechols to 0-quinones (catecholase activity), which is the initial step for the biosynthesis of melanin (Scheme 1) (7,13). 

Catechol and pyrogallol are good substrates for the mushroom enzyme. In crude preparations monophenols are also oxidized, but with a lag period. As purification proceeds the ability to oxidize monophenols is lost. The ability to oxidize monophenols was named cresolase activity by the Nelson school, as p-cresol was used as a representative substrate. 

The relation between oxidation of monophenols and polyphenols has been investigated extensively by Nelson and his students. The nature of the reactions studied is still not clear, but many interesting aspects have been explored. As in the case of other workers, it was found that there is a tendency for the ability to oxidize monophenols (cresolase) to be lost on purification, while polyphenol oxidation (catecholase) is more stable. The ratio of catecholase to cresolase activity of purified preparations appears to vary with electrophoretic mobility, and it has been suggested that a peptide component is lost from the enzyme, and that the purified preparations represent partially degraded enzyme. ... 

Tyrosinase is an enzyme complex (phenolase, polyphenol oxidase are other names which have been used for this enzyme), which catalyses of the ortho hydroxylation of monohydric phenols. The enzyme, which should not be confused with L-tyrosine hydroxylase mentioned above, contains Cu (I) and catalyses two distinct reactions—the hydroxylation of monohydric phenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to o-quinones (catecholase or catechol oxidase activity) . Most enzymes of this type, which are widely distributed in both the plant and animal kingdoms, exhibit both cataljrtic functions. Thus typically, the conversion of L-tyrosine (5) to L-dopa (15) and dopaquinone (36) which occurs in melanin biosynthesis is catalysed by an enzyme of the tyrosinase category. The two activities appear, in the majority of cases, to be functions of the same enzyme. However, certain o-diphenol oxidases such as those from tea , sweet potato and tobacco have been reported to show no capacity to catalyse the hydroxylation reaction but this is most probably due to destruction of the cresolase activity during purification. 

Fig. 11.12. Mode of action of grape tyrosinase on hydroxycinnamic acids (Mayer and Harel, 1979). (1) Cresolase activity (a coumaric acid, b cafeic acid) (2) Catecholase activity (a) cafeic acid (b) quinone...

The cresolase function of the phenolase complex requires a source of electrons. The oxidation of o-diphenols is thus required to maintain enzymic hydroxylation of monophenols (34,83,129,289,298,299, 486,517,571,586) but the reaction is also activated by other reducing agents (38,83,298,411,451,488,490). In the presence of purified py-rocatecholase, cresolase activity is inhibited (571). This inhibition can be reversed by addition of o-diphenol the relief lasts as long as diphenol is present. Furthermore, in the case of stereospecific mammalian phenolase complex, dihydroxyphenyl-L-alanine is a more efficient activating agent for hydroxylation of tyrosine than dihydroxy-phenyl-D-alanine (243). 

It is apparent that hypotheses proposed for the mechanism of action of the phenolase complex now comprise the reasonable permitted alternatives. Whichever is correct, one atom of the oxygen molecule consumed during hydroxylation of monophenols by the phenolase complex appears in the resulting o-diphenol and the other atom is reduced. The cresolase activity of this complex is accordingly a mixed function oxidase. The catecholase function, on the other hand, results in the reduction of both atoms of the oxygen molecule to water. It is therefore a four-electron transfer oxidase. 

Tyrosinase catalyzes the synthesis of melanin through the hydoxylation of tyrosine to dihydoxyphenylalanine (DOPA) and the subsequent oxidation of DOPA to dopaquinone. The unstable dopaquinone will polymerize and precipitate into melanin. However, in the presence of a reductor, the reaction will stop at the dlphenol level [2]. The cresolase activity of tyrosinase is of particular importance because it synthesizes DOPA. DOPA is a precursor of dopamine, an important neural message transmitter. 

---