Catechol oxidase, tyrosinase

Dioxygen activation is also accomplished at the dinuclear copper-active sites in tyrosinases and catechol oxidases. Tyrosinases (EC 1.14.18.1) are widely distributed throughout bacteria, fungi, plants, and animals, catalyzing the ortho-hydroxylation of phenols to catechols (phenolase activity. Equation (1)) and the oxidation of catechols to o-quinones (catecholase activity. Equation (2)). 

There has been enormous activity in the field of copper(I)-dioxygen chemistry in the last 25 years, with our information coming from both biochemical-biophysical studies and to a very important extent from coordination chemistry. This has resulted in the structural and spectroscopic characterization of a large number of copper dioxygen complexes, some of which are represented in Figure 14.2. The complex F, first characterized in a synthetic system was subsequently established to be present in oxy-haemocyanin, and is found in derivatives of tyrosinase and catechol oxidase, implying its involvement in aromatic hydroxylations in both enzymes and chemical systems. 

Catechol oxidase and tyrosinase are among the enzymes that have Type 3 dinuclear centres. However, the prototype of this class of proteins is the invertebrate oxygen transport protein, haemocyanin (Figure 14.5), for which structures of the oxy and deoxy forms have been determined at high resolution and confirm, as predicted from model compounds, that the... 

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. 

Hemocyanin [30,31], tyrosinase [32] and catechol oxidase (2) [33] comprise this class of proteins. Their active sites are very similar and contain a dicopper core in which both Cu ions are ligated by three N-bound histidine residues. All three proteins are capable of binding dioxygen reversibly at ambient conditions. However, whereas hemocyanin is responsible for O2 transport in certain mollusks and arthropods, catechol oxidase and tyrosinase are enzymes that have vital catalytic functions in a variety of natural systems, namely the oxidation of phenolic substrates to catechols (Scheme 1) (tyrosinase) and the oxidation of catechols to o-quinones (tyrosinase and catechol oxidase). Antiferromagnetic coupling of the two Cu ions in the oxy state of these metalloproteins leads to ESR-silent behavior. Structural insight from X-ray crystallography is now available for all three enzymes, but details... 

While only tyrosinase catalyzes the ortho-hydroxylation of phenol moieties, both tyrosinase and catechol oxidase mediate the subsequent oxidation of the resulting catechols to the corresponding quinones. Various mono- and dinu-clear copper coordination compounds have been investigated as biomimetic catalysts for catechol oxidation [21,194], in most cases using 3,5-di-tert-butylcatechol (DTBC) as the substrate (Eq. 16). The low redox potential of DTBC makes it easy to oxidize, and its bulky tert-butyl groups prevent un-... 

This class includes enzymes that use diphenols or related compounds as electron donors and oxygen as the acceptor, thereby forming the oxidized donor and water. Members include catechol oxidase (E.C. 1.10.3.1), laccase (E.C. 1.10.3.2), and o-aminophenol oxidase (E.C. 1.10.3.4). Laccase is also known as / -diphenoloxidase. whereas catechol oxidase is also known as diphenoloxidase, phenoloxidase, polyphenoloxidase, o-diphenolase, phenolase and tyrosinase. Many of these names are also used in reference to a different enzyme, monophenol monooxygenase (E.C. 1.14.18.1). This enzyme will be discussed further in Section 1.8.2.2. 

The interest in catechol oxidase, as well as in other copper proteins with the type 3 active site, is to a large extent due to their ability to process dioxygen from air at ambient conditions. While hemocyanin is an oxygen carrier in the hemolymph of some arthropods and mollusks, catechol oxidase and tyrosinase utilize it to perform the selective oxidation of organic substrates, for example, phenols and catechols. Therefore, establishment of structure-activity relationships for these enzymes and a complete elucidation of the mechanisms of enzymatic conversions through the development of synthetic models are expected to contribute greatly to the design of oxidation catalysts for potential industrial applications. 

Another fascinating topic which attracted the attention of many researchers is the difference in the enzymatic activities between catechol oxidase and the structurally related enzyme tyrosinase. Although both enzymes contain type 3 active... 

Co = Catechol oxidase He = Hemocyanin Tyr = Tyrosinase MePy2 = Al,Al-bis(2-pyridylethyl)methylamine TACN = l,3,5-triazocyclononane TMPA = Tris[(2-pyridyl) methyljamine XYL-O = Q ,Q -bis[Al,Al-bis(2-pyridylethyl) amino]-m-xylene-2-olate XYL = Q ,Q -bis[A, Al-bis(2-pyri-dylethyl)amino]-m-xylene XYL-R = Meta substituted a,a -bis[Al,Al-bis(2-pyridylethyl)amtno]-m-xylene N3 = N,N, Al -Tetrakis-(2-pyridin-2-yl-ethyl)-propane-1,2-diamine N4 = Al,Al,Al, Al -Tetrakis-(2-pyridin-2-yl-ethyl)-butane-1,2-diamine N5 = Al,Al,Al, Al -Tetrakis-(2-pyridin-2-yl-... 

Figure 1 Schematic representation of the dioxygen binding dinuclear copper enzymes Hemocyanin (He), Tyrosinase (Tyr), and Catechol oxidase (Co)...

This similarity in spectral properties implies that haemocyanins should also have catalytic activity. From the available body of experimental data, it is clear that the distinction between the two major functions — oxygen transport and enzymatic activity — is determined by the presence or absence of a protein domain covering the active site. In the case of tyrosinase and catechol oxidase, inactive pro-enzyme forms are activated by removal of an amino acid which blocks the entrance channel to the active site (indicated by the black bar in Figure 14.7). Haemocyanins behave as silent inactive enzymes but can be activated in the same way if the blocking amino acid is removed. In arthropods, like crabs, this is located in the N-terminal domain of a subunit whereas in molluscs, like octopus, it is in the C-terminal domain of a functional unit. 

Mushroom tyrosinase was extracted as described by Ingebrigtsen and Flurkey (J. Food Sci., in press). Tyrosinase activity was monitored using either catechol, dopa or tyrosine as the substrates. All assays were carried out in the presence and absence of 0.1% SDS (w/v) to detect active and latent enzyme activities. The catechol oxidase activity of tyrosinase was assayed in 50 mM phosphate (pH 6.0) containing 10 mM catechol and the absorbance monitored at 410 nm (25-26). The dopa oxidase activity of tyrosinase was assayed in 50 mM phosphate (pH 6.0) containing 5 mM L-dopa and the absorbance monitored at 475 nm. The tyrosine hydroxylase activity of tyrosinase was assayed in 33 mM phosphate (pH 6.0) containing 0.33 mM L-tyrosine and the absorbance monitored at 280 nm. Protein content was determined by the method of Lowry et al. (26). 

The type-3 copper proteins hemocyanin, tyrosinase, and catechol oxidase with dicopper active sites and three histidine imidazole donors per copper... 

Tyrosinase is classified into the type-3 copper protein family, as are catechol oxidase and the respiratory pigment hemocyanin. During the catalytic reaction, the type-3 copper center of tyrosinase exists in three redox forms. The deoxy form (Cu(I)-Cu(I)) is a reduced species, which binds oxygen to give the oxy form (Cu(II)-02 -Cu(II)). In the oxy form, molecular oxygen is bound as peroxide in a side-on bridging mode, which destabilizes the... 

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