Catecholase reactions

Such a mechanism accounts for the overall stoichiometry of four electrons passed in pairs to molecular oxygen. This stoichiometry must also be preserved in the catecholase reaction see below). The mechanism also requires a second cosubstrate binding site for the activating catechol. A plot of 1/tiag s. DOPA concentration (Figure 13) shows... 

Since one molecule of oxygen ultimately oxidizes two molecules of reducing substrate, the complete mechanism for the catecholase reaction must also involve two catechol molecules even if they are only sequentially oxidized. However, it might be that the activator site is also involved in the catecholase reaction and that the oxidations by tyrosinase involve a complex between two substrate molecules, copper, and oxygen, to allow for a simultaneous transfer of two pairs or four electrons. 

Michaelis-Menten saturation kinetics should occur only at low substrate concentrations, near 10 M. Using a spectrophotometric assay it is possible to observe statistically significant deviations as shown by the Line-weaver-Burke and Eadie plots in Figure 14 using DOPA as the substrate in the catecholase reaction. 

As compared to the oxygenation reaction of phenols to catechols (phenolase reaction), dehydrogenation of catechols to the corresponding o-quinones (catecholase reaction) proceeds more readily. Thus, the catalytic activity of several tyrosinase and catechol oxidase models have been examined using 2,4-di-tert-butylcatechol (DTBC) as a substrate.Direct reactions between the (/r-77 77 -peroxo)dicopper(II) complexes and DTBC also have been studied at a low tempera-and a semiquinone-copper(II) complex has been isolated and structurally characterized... 

The catecholase reaction 9 is energetically simpler than the pheno-lase reaction 8 and is therefore promoted by several copper(II) complexes in... 

Recently, the activation and thermodynamic parameters for the catalytic DTBCH2 oxidation promoted by [Cu2(L55)]" in a mixed aqueous/organic, cryogenic solvent have been obtained (157). The catecholase reaction proceeds also in this case with a biphasic rate of DTBQ development, and the rates of the two steps still exhibit hyperbolic dependence on DTBCH2 concentration. However, the second step under these conditions also depends linearly on O2 concentration. The spectroscopic and kinetic analysis of the system allow the formulation of the following mechanism ... 

Key words Catalytic oxidation, homogeneous catalysis, dioxygen activation, dioxygen conplexes, biomimetic oxidation, functional metaUoenzyme models, oxidation mechanisms, oxidative dehydrogenation, oxygen insertion, aUcene epoxidation, catecholase reaction... 

The simplest, but least accurate, method of assaying DPO activity is to record the final color yield when the enzyme is incubated with a suitable chromogenic substrate such as catechol, DOPA, or 4-methylcatechol. DOPA is the most frequently used substrate in colorimetric assays because it yields a dark brown/black end-product. In this reaction, catecholase catalyzes the conversion of DOPA to dopaquinone and then to the red dopachrome, which subsequently polymerizes to yield dark brown melanin-type pigments. Unfortunately, this simple procedure has serious limitations, as it measures the end-product of a sequence of reactions rather than the true initial reaction rate. Furthermore, because different substrates yield different final colors, valid kinetic comparisons between substrates are not possible. Nevertheless, this simple assay technique has proved adequate for useful comparative studies of the levels of enzymic browning in different fruit varieties and similar problems (Vamos-Vigyazo, 1981 Machiex et al., 1990). 

Despite its tetranuclear structure in the solid state, the dicopper(II) complex was found to dissociate in solution into dinuclear units at the concentration levels used for catecholase activity studies. Similarly to the copper(II) complex with the ligand [22]py4pz, the present complex also catalyzes the oxidation of the model substrate DTBCH2 in methanol. However, several unexpected observations have been made in the present case. First, the rate-determining step in the catalytic reaction was found to change with the substrate-to-complex ratio. Thus, at low substrate-to-... 

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. 

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 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]. 

Alternative reaction mechanisms include a radical mechanism proposed by Kitajima and Morooka and a mechanism involving a Cu(III) intermediate based on measurements of model compounds. On the basis of the crystal structure of the catechol oxidase-PTU inhibitor complex, monodentate binding of the substrate was suggested for catechol oxidase. A radical mechanism, as proposed for the weak catecholase activity found in Octopus vulgaris hemocyanin, is also possible for catechol oxidase due to the strong structural relationship between catechol oxidase from /. batatas and odg hemocyanin as described above. 

In this chapter, the dioxygen activation mechanism at the dinuclear copper-active sites of tyrosinase and catechol oxidase has been surveyed. In both enzymes, a (ji-rfirf -peToxo) dicopper(II) complex has been detected and characterized as a common reactive intermediate by several spectroscopic methods. In spite of longstanding efforts in the enzymological studies, mechanistic details of the enzymatic reactions (phenolase and catecholase activities) still remain ambiguous. On the other hand, recent developments in the model chemistry have provided a great deal of information about the structure and physicochemical properties as well as the reactivity of the peroxo intermediate and have advanced our understanding of the enzymatic reactions. 

X 10 s , respectively), indicating that the reaction of the peroxo species is rate limiting. The process in fact corresponds to one catecholase-like cycle and occurs with a stoichiometry of 2 catechohl O2 (reaction 9 and Scheme 2). No dimers arising from C-C coupling, or C-0 coupling reactions were detected in the oxidation of both substrates. 

FIGURE 11.3 Comparison of reactions catalyzed by catecholase (o-DPO) and laccase (p-DPO). (From Walker, J.R.L., Enzymatic browning in frnits Its biochemistry and control, in Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R., Eds., American Chemical Society, Washington, DC, 1995. With permission.)... 

Most PPO preparations from potato, apple, mushroom, and bean possess both monophenol and diphenol oxidase activities, whereas those from tea leaf, tobacco, mango, banana, pear, peach, and sweet cherry have been reported not to act on monohydroxyphenols [28]. Whether a single enzyme system exhibits both mono- and diphenol oxidase activities is still unclear. It was suggested that both cresolase and catecholase functions are catalyzed by a single site. Verdedoncella apple PPO showed both monophenol and diphenol oxidase activities with a reaction mechanism involving one... 

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 unique property of this enzyme is that the product of the first monoxygenation step, o-diphenol, serves as the electron donor for the reduction of the cupric ions with formation of the corresponding o-quinone 161, 274). The products formed by catecholase activity from tyrosine are extremely reactive and undergo intermolecular reactions to form indole derivatives which subsequently polymerize to melanin. 

As mentioned in Section 2, incorporation of catalysts in porous matrices not only presents the advantages of heterogeneous catalysts (easy separation of the catalyst from the reaction mixture) but also prevents deactivation pathways. This is demonstrated with the example of dicopper acetate dimers incorporated in various mesoporous inorganic matrices. Tested for phenolase and catecholase activity, they displayed a 10-fold increase of the TON compared to neat copper acetate, which was ascribed to the isolation of the active site. Chemoselectivity (hydroxyaryls vs other aryls) and regio selectivity (phenol ortho hydroxylation) have been reported with this system. ... 

Figure 32 (a) Catalytic reactions performed by calix[6]-tris(pyridine)Cu(II) via the peroxide shunt pathway. (b) Catecholase... 

Tyrosinase (E.C. 1.14.18.1, monophenol monooxygenase) is a copper monooxygenases enzyme that catalyzes two different oxygen-dependent reactions, namely the oxidations of both monophenols (cresolase or monophenolase activity) and o-diphenols (catecholase or diphenolase activity) into reactive o-quinones [28, 89]. 

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. ... 

Polyphenol oxidase catalyzes two reactions first the hydroxylation of a monophenol to o-diphenol (EC 1.14.18.1, monophenol monooxygenase) followed by an oxidation to o-quinone (EC 1.10.3.1, o-diphenol oxygen oxidoreductase). Both activities are also known as cresolase and catecholase activity. At its active site, polyphenol oxidase contains two Cu ions with two histidine residues each in the ligand field. In an ordered mechanism (cf. 2.5.1.2.1) the enzyme first binds oxygen and later monophenol with participation of the intermediates shown in Fig. 2.8. The Cu ions change their valency (Cu Cu ). The newly formed complex ([] in Fig. 2.8) has a strongly polarized... 

Flqure 9.31 Reactions catalysed by polyphenol oxidases with creasolase, catecholase and laccase activities. 

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. 

---