Tyrosinase active site

Ross, P. K., and E. I. Solomon. 1991. An Electronic Structural Comparison of Cooper-Peroxide Complexes of Relevance to Hemocyanin and Tyrosinase Active Sites. J. Am. Chem. Soc. 113, 3246. 

Now that the appropriate enzyme level has been determined, the kinetic constants may be evaluated. The Ku for L-dopa can be obtained by setting up the same assay as in part B, except that the factor to vary will be the concentration of L-dopa. The concentration of L-dopa in part B was sufficient to saturate all the tyrosinase active sites, so the rate depended only on the enzyme concentration. In part C, L-dopa levels will be varied over a range that is nonsaturating. 

A series of hemocyanin and tyrosinase active site derivatives (Fig. 23) can be prepared61"66), allowing systematic variation of the binuclear copper active site and chemical perturbation for spectral studies. In the simplest derivative, met-apo, one copper has been removed and the remaining copper oxidized to the spectroscopically accessible Cu(II). Next in complexity is a mixed-valent binuclear copper site. The Cu(II), in this half-met derivative, exhibits open-shell d9 spectroscopic features and the Cu(I), though spectroscopically inaccessible, can still be studied by comparison to the met-apo derivative. Two derivatives have formally binuclear cupric sites met, which is EPR-non-detect-able, and dimer, which exhibits an intense broad EPR signal. Spectroscopic study of these derivatives has led to the present picture of the coupled binuclear copper protein active site shown at the bottom of Fig. 23. 

Recently, it was foimd that kojic add-tripeptide amides showed similar tyrosinase inhibitoiy activities to those of kojic add-tripeptide free adds but exhibited superior storage stability than those of kojic acid and kojic add-trip>eptide free acids (Noh, 2007). To find further kojic acid derivatives with higher tyrosinase inhibitory activity, stability, and synthetic effidency, a library of kojic add-amino acid amides (KA-AA-NH2) prepared and screened for their tyrosinase inhibitoiy activities. It was also confirmed that the kojic add-phenylalanine amides reduced the amount of dopachrome production during the melanin formation. It was suggested that a tyrosinase inhibition mechanism of KA-AA-NH2 based on the possible hydrophobic interadions between the side chain of KA-AA-NH2 and tyrosinase active site by a docking program (Noh, 2009 Kim, 2004). 

Since the oxidative polymerization of phenols is the industrial process used to produce poly(phenyleneoxide)s (Scheme 4), the application of polymer catalysts may well be of interest. Furthermore, enzymic, oxidative polymerization of phenols is an important pathway in biosynthesis. For example, black pigment of animal kingdom "melanin" is the polymeric product of 2,6-dihydroxyindole which is the oxidative product of tyrosine, catalyzed by copper enzyme "tyrosinase". In plants "lignin" is the natural polymer of phenols, such as coniferyl alcohol 2 and sinapyl alcohol 3. Tyrosinase contains four Cu ions in cataly-tically active site which are considered to act cooperatively. These Cu ions are presumed to be surrounded by the non-polar apoprotein, and their reactivities in substitution and redox reactions are controlled by the environmental protein. 

Tyrosinase is a monooxygenase which catalyzes the incorporation of one oxygen atom from dioxygen into phenols and further oxidizes the catechols formed to o-quinones (oxidase action). A comparison of spectral (EPR, electronic absorption, CD, and resonance Raman) properties of oxy-tyrosinase and its derivatives with those of oxy-Hc establishes a close similarity of the active site structures in these proteins (26-29). Thus, it seems likely that there is a close relationship between the binding of dioxygen and the ability to "activate" it for reaction and incoiporation into organic substrates. Other important copper monooxygenases which are however of lesser relevance to the model studies discussed below include dopamine p-hydroxylase (16,30) and a recently described copper-dependent phenylalanine hydroxylase (31). 

Evidence tom a variety of sources indicates that the active site of tyrosinase is very similar to that of hemocyanin, a dioxygen-binding protein found in molluscs and arthropods (15,16). This type of active site contains two copper ions, which are cuprous in the deoxy state, and which reversibly bind dioxygen, forming the oxy form of the enzyme or protein in which a peroxy ligand bridges between two cupric ions. 

A coupled binuclear copper active site is found in a variety of different metalloprotelns Involved in dloxygen reactions. These Include hemocyanln (reversible O- binding), tyrosinase (O2 activation and... 

Our earlier research on the coupled binuclear copper proteins generated a series of protein derivatives in which the active site was systematically varied and subjected to a variety of spectroscopic probes. These studies developed a Spectroscopically Effective Model for the oxyhemocyanin active slte.(l) The coupled binuclear copper active site in tyrosinase was farther shown to be extremely similar to that of the hemocyanlns with differences in reactivity correlating to active site accessibility, and to the monophenol coordinating directly to the copper(II) of the oxytyroslnase site.(2) These studies have been presented in a number of reviews.(3) In the first part of this chapter, we summarize some of our more recent results related to the unique spectral features of oxyhemocyanin, and use... 

Figure 7. The Spectroscopically Effective Active Site of hemocyanln and tyrosinase.

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... 

Many metalloproteins contain more than one metal center. Tyrosinase, for example, has a dinuclear Type 3 (T3) copper active site which, in its oxidized form, comprises two Cu(II) centers each held by three histidine groups with a p-r 2 r 2 peroxido bridging ligand (Fig. 22). 

Fig. 22. Schematic representation of the oxidized T3 active site in Tyrosinase...

Soluble tyrosinase (sTr) itself crystallizes with a caddie protein, ORF378 (the green ribbon in Fig. 26), which was removed in the LFMD simulations. As also shown in Fig. 26, this does not have a significant effect on the protein backbone. However, the absence of ORF378 in the simulations leads to some significant local variations in the active site structure. 

One of the questions surrounding the mechanism of tyrosinase concerns the initial site of attack. As a control, LFMD simulations of a model for the sTy active site, Meim6 (Fig. 28), give identical behavior for each Cu center consistent with its symmetry. In contrast, the LFMD simulations clearly distinguish the two copper sites in the sTy enzyme which must result from the protein environment (Fig. 29). 

Polyphenol oxidase (PPO) (EC 1.14.18.1 monophenol monooxygenase [tyrosinase] or EC 1.10.3.2 0-diphenol 02-oxidoreductase) is one of the more important enzymes involved in the formation of black tea polyphenols. The enzyme is a metallo-protein thought to contain a binudear copper active site. The substance PPO is an oligomeric particulate protein thought to be bound to the plant membranes. The bound form of the enzyme is latent and activation is likely to be dependent upon solubilization of the protein (35). PPO is distributed throughout the plant (35) and is localized within in the mitochondria (36), the cholorplasts (37), and the peroxisomes (38). Using antibody techniques, polyphenol oxidase activity has also been localized in the epidermis palisade cells (39). Reviews on the subject of PPO are available (40—42). 

Figure 2.22. Initial velocity of oxygen consumption as a function of the substrate (catechol) concentration in the presence of 0.074mg (7.11 x 1(T9M) tyrosinase (A), 2.0mg (2.8 x 1(T4M with a corresponding concentration of the mineral active sites, [M5]1.71xl(r6) of 8-Mn02 (B) and 10.0 mg (1.40 x 10 3M with a corresponding concentration of the mineral active sites, [M ]8.54 x 10-6) of S-Mn02 (C). Reprinted from Naidja, A., Liu, C., and Huang, P. M. (2002). Formation of protein-birnessite complex XRD, FTIR, and AFM analysis. J. Coll. Interface Sci. 251,46-56, with permission from Elsevier.

As the focus of this review is on copper-dioxygen chemistry, we shall briefly summarize major aspects of the active site chemistry of those proteins involved in 02 processing. The active site structure and chemistry of hemocyanin (He, 02 carrier) and tyrosinase (Tyr, monooxygenase) will be emphasized, since the chemical studies described herein are most relevant to their function. The major classes of these proteins and their origins, primary functions, and leading references are provided in Table 1. Other classes of copper proteins not included here are blue electron carriers [13], copper-thiolate proteins (metallothioneines) [17], and NO reductases (e.g., nitrite [NIR] [18] or nitrous oxide [19]). 

---