Polyphenol oxidase oxidation mechanism

Hutcheson SW, Buchanan BB. Polyphenol oxidation by Viciafaba chloroplast membranes studies on the latent membrane-bound polyphenol oxidase and on the mechanism of photochemical polyphenol oxidation. Plant Physiol 1966 66 1150-1154. 

RP-HPLC methods have been frequently applied for the investigation of various chemical, biochemical and biophysical processes in in vitro model systems. Thus, the separation of new compounds achieved by enzymatic oxidation of phloridzin was carried out by semi-preparative RP-HPLC. Phloridzin was incubated with a polyphenol oxidase prepared from apple pulp for 6h at 30°C under air agitation. After incubation the suspension was filtered, stabilized by NaF and injected into the RP-HPLC column using diluted acetic acid-ACN gradient. The new compounds were isolated and identified by NMR and MA techniques. The proposed mechanism of the formation of new phloridzin derivatives 3 and 4 is shown in Fig. 2.159. The results illustrate that RP-HPLC can be successfully used for the study of enzymatic processes in model systems [331],... 

The aerobic oxidation of RAA (Figure 1) occurs rapidly when metal catalysts, particularly copper or iron, or enzymes such as ascorbic acid oxidase, polyphenol oxidase, peroxidase, and cytochrome oxidase are present. The anaerobic destruction of ascorbic acid may proceed by a variety of mechanisms that have been postulated (2,3) but not verified. 

Ascorbic acid browning is also inhibited by the addition of sulfite (Wedzicha and McWeeny, 1974). The same holds for polyphenol oxidase-catalyzed oxidation of natural phenols in fruit. The mechanism of the inhibition is by reaction of oquinone intermediates with sulfite, which leads to nonreactive sulfocatechols (Wedzicha, 1995). 

Further validation of the mechanism proposed for the catecholase activity of the dicopper complexes [Cu2(L66)]" , [Cu2(L55)], and [Cu2(EBA)]" " (Scheme 17) has been obtained investigating the inhibitory effect of kojic acid [5-hydroxy-2-(hydroxymethyl)-y-pyrone] (154). This fungal metabolite is one of the most efficient inhibitors of mushroom tyrosinase and other polyphenol oxidases (160,161). When the catalytic oxidation of DTBCH2 was studied in the presence of kojic acid, strong competitive inhibition was observed in the steps exhibiting substrate concentration dependence,... 

FIGURE 11.6 Proposed kinetic scheme depicting the mechanisms of oxidation of o-diphenol (catechol top a) and monophenol (phenol bottom b) for the N. crassa polyphenol oxidase. (From Whitaker, J.R. and Lee, C.Y., Recent advances in chemistry of enzymatic browning, in Enzymatic Browning and Its Prevention, Lee, C.Y. and Whitaker, J.R., Eds., American Chemical Society, Washington, E)C, 1995, 3. With permission.)... 

Oxidations now known to be catalyzed by copper-containing enzymes were noticed over a century ago, when Schoenbein observed that oxidation of natural substrates resulted in pigment formation in mushrooms. Individual enzymes were gradually identified laccase by Yoshida in 1883 and tyrosinase by Bertrand in 1896. However, it was not imtil potato polyphenol oxidase was isolated in 1937 by Kubowitz that the role of copper was defined. The family of copper oxidases includes a number of enzymes of both plant and animal origin that may very probably be found to react through similar mechanisms, but which exhibit a number of individual characteristics. The enzymes to be described in this section include potato phenol oxidase, mushroom polyphenol oxidase (tyrosinase), laccase, mammalian and insect tyrosinase, and ascorbic acid oxidase. Each of these differs in certain respects from the others, and undoubtedly other related enzymes will be described from other sources that resemble these, but also display individualities. In these cases, identities in nomenclature must not be extended to imply identities in enzyme structure or activity. 

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... 

NADPH oxidase activity in circulating neutrophils (unpublished observations). NADPH oxidase-dependent superoxide production appears to be abnormally high in mononuclear cells from these patients (Formno et al. 2005). The absence of a commercial pharmacological treatment to reduce or inhibit systemic NADPH oxidase enzyme complex highlights the importance of our results, which demonstrate for the first time that the oxidative stress produced by systemic NADPH oxidase may be counteracted by supplementation with polyphenols. Our in vitro studies suggest that the mechanism involved in this effect is the reduction of p22phox, p47phox, and NOX expression (unpublished observations). 

The lag time effect probably results from the inhibition of copper-containing oxidases and other copper-catalyzed oxidative processes in apple by Sporix. These oxidative reactions normally would bring about the rapid loss of AA and permit browning to occur once the added AA was depleted (18). Sporix also would inhibit PPO directly by chelation of its copper (3), thereby decreasing the rate of polyphenol oxidation and subsequent browning. The ability of Sporix to exert its effect on enzymatic browning by these two independent mechanisms probably accounts for the apparent synergism obtained with Sporix-AA combinations. 

Multicopper oxidases are typically active in the catalytic one-electron oxidation of a variety of diphenolic, polyphenolic, enediolic, and aminophe-nolic substrates 1,53,166,167). The mechanism of these reactions is complex and, as discussed in Section I, it involves a sequence of four one-electron oxidations of substrate molecules. The radical products of these reactions undergo dismutation, as shown in Scheme 21 for the oxidation of ascorbate to semidehydroascorbate radical 168,169). The substrate binds to the enzymes close to type 1 Cu, whereas the trinuclear cluster is only accessible to dioxygen, or other small molecules. This situation is clearly difficult to reproduce in a model system and for this reason the type of model oxidation reactions that have been studied so far using synthetic trinuclear copper complexes is more related to the activity of type 3 Cu enzymes than multicopper oxidases. Nevertheless, such trinuclear complexes open new perspectives in stereoselective catalysis, because one of the metal centers... 

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