Catecholase

Krebs and co-workers synthesized a series of dinuclear copper(II) complexes as models for catechol oxidase 91 (365) (distorted SP Cu-Cu 2.902 A), (366) (distorted five-coordinate geometry Cu-Cu 3.002A), (367) (distorted SP Cu-Cu 2.995 A), (368) (distorted five-coordinate geometry Cu-Cu 2.938 A), and (369) (distorted SP Cu-Cu 2.874 A). These complexes were characterized by spectroscopic and electrochemical methods. From kinetic analysis, a catalytic order for catecholase activity (aerial oxidation of 3,5 -di - ter t-buty lcatec h o 1) was obtained.326... 

Cabanes J, Chazarra S and Garcia-Carmona F. 1994. Kojic acid, a cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase. J Pharm Pharmacol 46(12) 982—985. 

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. 

Alternate Protocol 1 Spectrophotometric Assay of Catecholase Activity C4.1.7... 

The diphenol oxidases (DPOs) includes two major groups of enzymes the ortho-DPOs (also known as catecholases, polyphenol oxidases, and tyrosinases) and the para-DPOs (more usually known as laccases). The names catecholase and laccase are used in this unit. 

Substrates should be prepared fresh daily and discarded if they show any signs of discoloration due to autooxidation. 4-Methylcatechol usually gives the highest rate of 02 uptake in catecholase assays, and toluquinol commonly gives the highest rate for the assay oflaccases. It is important that the substrate be added last. 

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). 

The most common naturally occurring substrates for catecholases in plants include chlorogenic acid, catechin, DOPA, and dopamine this topic is covered in detail in the literature (see Key References). In most plants and fungi, some natural substrate(s) will be present in the tissue to be analyzed (Figure C4.1.4). 

Catecholases and laccases may be differentiated by the use of substrate specificity tests and selective specific inhibitors (Walker and McCallion, 1980 Ferrar and Walker, 1996 Table C4.1.1). Salicylhydroxamic acid (SHAM), PVP, and/or cinnamic acids (cinnamic, p-coumaric, or ferulic) are probably the best choice for catecholase inhibitors, whereas cetyltrimethylammonium bromide (CETAB) has been found to inhibit most laccases. 

Table C4.1.1 Differentiation of Catecholase and Laccase by Use of Selective Substrates and Inhibitors...

Comprehensive review of the role of catecholases and laccases in higher plants and fungi. 

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... 

These controversial findings inspired numerous subsequent studies on the structure-activity relationship of catalytically active compounds. Very detailed mechanistic studies on the catecholase activity of a series of structurally related dicopper(II) complexes have also been published by Casella and co-workers [37-40], who have grouped together different mechanisms earlier proposed for the catecholase activity of dicopper(II) complexes, as shown in Scheme 5.2. 

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-... 

Traverson-Rueda, S. Singleton, V.L. Catecholase Activity in Grape Juice and... 

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 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. 

The subsequent catecholase activity of tyrosinase requires easy removal of electrons from the phenolic oxygens at the 1 and 2 positions... 

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-... 

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. 

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