Tyrosinase model systems

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

Figure 10 A tyrosinase model system reversible oxygenation of xylyl dicopper(I) complex 10 to give 11, followed by hydroxylation to give 12.

The xylyl hydroxylating system appears to possess a number of elements seen in enzyme catalyzed reactions, and the presence of a dicopper moiety, which effects a specific aromatic ring hydroxylation, identifies it as a tyrosinase model system. Analogous to the enzyme, either dieopper(I)/02 or met dicopper(II)/ H202 reactions result in xylyl hydroxylation. Although not yet proved absolutely, [Cu2(R—XYL—Y)(02)]2+ (11) is suggested to possess a p.-ii2 Ti2-022 structure, the one now favored for oxy-Hc and oxy-Tyr. Very likely, it acts as an electro-... 

The properties of Kitajima s p-p2 Ti2-pcroxo dicopper(II) complex lead to the conclusion that this is the likely structure in oxyhemocyanin and oxytyrosi-nase this is perhaps the most important contribution from this type of model chemistry. A distorted or closely related peroxo-dicopper(II) species appears to be involved in aromatic hydroxylation proceeding in a well-characterized tyrosinase model system. 

We have studied the reaction between lysine and polyphenols in a model system containing casein and caffeic acid, incubated at room temperature at pH 7 and 10 with and without tyrosinase. In the absence of tyrosinase and oxygen, reactive lysine does not change in the absence of tyrosinase and in the presence of oxygen, reactive lysine decreases when the pH is higher than 9. In the presence of tyrosinase and oxygen, the level of reactive lysine decreases when the pH is 7, which is the optimum pH of the enzymic activity. At pH 7, with tyrosinase, the reaction rate is low, while at pH 10, the reaction rate is more rapid (119). 

Activation mechanism. What happens during activation in hemocyanins and how can we use them as model systems to understand tyrosinase/catecholoxidase activity Arthropod and mollusc hemocyanins have to be considered separately, although the processes are similar. 

Other DFT-B3LYP calculations were performed by Siegbahn and coworkers [343], this time on a neutral model system L3CU. .. Cu L3 to probe the mechanism of tyrosinase action. The ligands L chosen to model histidines, were either ammonia or formimine. The authors focus on the choice of chemical model and its limitations, the location of the transition state for 0-0 activation... 

Enzymes and proteins containing binuclear copper centers for the utilization of oxygen in organisms have attracted a great deal of attention in recent years. These proteins play critical roles in transport (hemocyanin), neurotransmitter synthesis (dopamine-P-hydroxylase),2 and pigment synthesis (tyrosinase), among other functions. Studies on natural systems as well as on model systems have led to further insight into the processes... 

There has been considerable interest in designing and testing model systems that mimic the action of tyrosinases. This effort is motivated by the need of understanding enzymatic oxidations and by the possible emergence of simple catalytic systems for the hydroxylation of arenes (as well as alkanes). 

Catalytic mechanisms for tyrosinase activity have been proposed (7) based on both biochemical and model system studies (see later). The former include key recent kinetic studies from Itoh (50) and Casella (51). Mechanistic details have also been suggested based on computational studies (52,53). (Scheme 3) shows a well-accepted proposed mechanism, which includes phenolase and catecholase cycles. Here, tyrosinase is present in three redox forms during the cycle. The reduced deoxy Cu(I)-Cu(I) entity binds O2 to generate the oxyform Cu(II)-02 -Cu(II) in which the peroxide group occurs in a side-on -binding mode the... 

The chemical and enzymatic browning reactions of plant polyphenols and their effects on amino acids and proteins are reviewed. A model system of casein and oxidizing caffeic acid has been studied in more detail. The effects of pH, time, caffeic acid level and the presence or not of tyrosinase on the decrease of FDNB-reactive lysine are described. The chemical loss of lysine, methionine and tryptophan and the change in the bioavailability of these amino acids to rats has been evaluated in two systems pH 7.0 with tyrosinase and pH 10.0 without tyrosinase. At pH 10.0, reactive lysine was more reduced. At pH 7.0 plus tyrosinase methionine was more extensively oxidized to its sulphoxide. Tryptophan was not chemically reduced under either condition. At pH 10.0 there was a decrease in the protein digestibility which was responsible for a corresponding reduction in tryptophan availability and partly responsible for lower methionine availability. Metabolic transit of casein labelled with tritiated lysine treated under the same conditions indicated that the lower lysine availability in rats was due to a lower digestibility of the lysine-caffeoquinone complexes. 

While these studies show that both the Li-Ti Ti -peroxodicopper(II) and the bis-p,-oxO dicopper(III) cores are capable of hydroxylating arenes in model systems, the question still remains of whether the enzyme tyrosinase hydroxylates its substrates directly with its observed peroxodicopper(II) core or if 0-0 bond breaking occurs first before C-H bond activation. 

Oxygenation rates were first examined for the system [Cu (R-XYL-H)]2+. In the model described here, two bis [2-(2-pyridyl)ethyl] amine (PY2) units are linked by a xylyl spacer group (R = H). Although initially proposed as a crude hemocyanin model, this system now is studied as a model for tyrosinase and is an example of hydrocarbon oxygenation taking place under mild conditions—that is, < 1 atm 02... 

Fukuzumi and co-workers described spectroscopic evidence for a ix-rf- ] -peroxo-(Cu )2 species stabilized with a fcidentate nitrogen ligand, but no (catalytic) oxidation behavior towards catechol was noted (a related trinu-clear copper species converted 2,4-di-ferf-butylphenol stoichiometrically towards the biphenol derivative) [224], Stack et al. have described a similar ] -peroxo-(Cu )2 species (28, vide supra) that could be considered a structural and functional model for tyrosinase-activity, as it efficiently reacted with catechol, benzyl alcohol and benzylamine to yield quinone (95%), benzaldehyde (80%) and benzonitrile (70%) [172,173]. This dinuclear per-0X0 species is generated by association of two monomeric copper centers, in contrast to the systems based on dinucleating Ugand scaffolds described above. 

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