Oxidative enzymes peroxidases

An interesting further example of a homolytic aromatic reaction involves the oxidation of phenols in basic solution with one-electron oxidising agents (e.g. Fe(m), H20 + the enzyme peroxidase) ... 

Although the exact mechanism of degradation at metabolic level for each compound or group of compounds is not well known, the involvement of extracellular oxidative enzymes such as LAC, MnP, LiP, and versatile peroxidase (VP) (see Tables 1 and 2 of Chap. 6) and intracellular monooxygenases as cytochrome P-450 is well documented for pollutants such as hydrocarbons, dyes, and halogenated solvents [25]. To determine the actual role of the extracellular enzymes, many studies are performed in vitro experiments with purified enzymes. In the case of cytochrome P-450, usually inhibitors are used. 

It is important to compare the catalytic properties of Prussian blue with known hydrogen peroxide transducers. Table 13.2 presents the catalytic parameters, which are of major importance for analytical chemistry selectivity and catalytic activity. It is seen that platinum, which is still considered as the universal transducer, possesses rather low catalytic activity in both H202 oxidation and reduction. Moreover, it is nearly impossible to measure hydrogen peroxide by its reduction on platinum, because the rate of oxygen reduction is ten times higher. The situation is drastically improved in case of enzyme peroxidase electrodes. However, the absolute records of both catalytic activity... 

The monophenolase activity of PPO is generally defined as the first step in the melaniza-tion pathway and consists of the o-hydroxylation of the monophenol to odiphenol. This activity distinguishes PPO from other phenol-oxidizing enzymes, such as laccase and peroxidase, and is characterized by the following facts ... 

If step III in Scheme 6 is ignored, a simplification is reached that has been demonstrated to be reasonable with certain substrates (see below). As a consequence, then the 1 catalysts should oxidize targeted reductants in accordance with Eq. (13) (here [Fe-TAML] is the total concentration of all TAML iron in solution). Eq. (13) implies that the catalysis by 1 mimics the steady-state oxidation by peroxidase enzymes, where Compound I is much more reactive than Compound II (55). 

Several compounds can be oxidized by peroxidases by a free radical mechanism. Among various substrates of peroxidases, L-tyrosine attracts a great interest as an important phenolic compound containing at 100 200 pmol 1 1 in plasma and cells, which can be involved in lipid and protein oxidation. In 1980, Ralston and Dunford [187] have shown that HRP Compound II oxidizes L-tyrosine and 3,5-diiodo-L-tyrosine with pH-dependent reaction rates. Ohtaki et al. [188] measured the rate constants for the reactions of hog thyroid peroxidase Compounds I and II with L-tyrosine (Table 22.1) and showed that Compound I was reduced directly to ferric enzyme. Thus, in this case the reaction of Compound I with L-tyrosine proceeds by two-electron mechanism. In subsequent work these authors have shown [189] that at physiological pH TPO catalyzed the two-electron oxidation not only L-tyrosine but also D-tyrosine, A -acetyltyrosinamide, and monoiodotyrosine, whereas diiodotyrosine was oxidized by a one-electron mechanism. 

Two additional systems were exploited in order to confirm the involvement of free-radical processes during vindoline oxidations. These were the enzyme peroxidase and photochemistry. Horseradish peroxidase (HRP) oxidized both vindoline and 16-O-acetylvindoline in the presence of hydrogen peroxide. Vindoline was converted to the enamine dimer 59 (78). During the reaction, the following sequence of redox reactions occurs ... 

In recent years, numerous applications of such peroxidase-catalyzed oxidative coupling of phenols and aromatic amines have been reported (Table 7). These peroxidase-catalyzed biotransformations lead to modified natural products with high biological activities [110-118]. Several examples have also been described for the oxidative coupling of phenols with peroxidases and other oxidative enzymes from a variety of fungal and plant sources as whole cell systems... 

Optically active epoxides are important building blocks in asymmetric synthesis of natural products and biologically active compounds. Therefore, enantio-selective epoxidation of olefins has been a subject of intensive research in the last years. The Sharpless [56] and Jacobsen [129] epoxidations are, to date, the most efficient metal-catalyzed asymmetric oxidation of olefins with broad synthetic scope. Oxidative enzymes have also been successfully utilized for the synthesis of optically active epoxides. Among the peroxidases, only CPO accepts a broad spectrum of olefinic substrates for enantioselective epoxidation (Eq. 6), as shown in Table 8. 

Enzymatic browning. Phenol-oxidizing enzymes (such as tyrosinase and peroxidase) oxidize tyrosine residues into reactive quinone derivatives, which will condense into colored polymers (melanins). Melanins are rich in carboxyl groups and therefore have high affinity for divalent metal ions such as calcium. 

There are a few common enzymes that have been employed in these types of assay systems over the years, the chief among them being the peroxidase enzyme (3). Peroxidase has an oxidative function when in conjunction with a source of oxygen, transferring electrons to a molecule, which becomes oxidized. The peroxidase enzyme found in the horseradish plant has been used for its ability to carry out this function, for the fact that it is easily obtained, and for the antigenic differences from most mammalian forms of the enzyme. The oxidative function of this enzyme allows for the use of chromogens, which when oxidized, not only change color, but precipitate in such a manner as to render a permanent preparation. 

The detailed mechanism of P aeruginosa CCP has been studied by a combination of stopped-flow spectroscopy (64, 65, 84, 85) and paramagnetic spectroscopies (51, 74). These data have been combined by Foote and colleagues (62) to yield a quantitative scheme that describes the activation process and reaction cycle. A version of this scheme, which involves four spectroscopically distinct intermediates, is shown in Fig. 10. In this scheme the resting oxidized enzyme (structure in Section III,B) reacts with 1 equiv of an electron donor (Cu(I) azurin) to yield the active mixed-valence (half-reduced) state. The active MV form reacts productively with substrate, hydrogen peroxide, to yield compound I. Compound I reacts sequentially with two further equivalents of Cu(I) azurin to complete the reduction of peroxide (compound II) before returning the enzyme to the MV state. A further state, compound 0, that has not been shown experimentally but would precede compound I formation is proposed in order to facilitate comparison with other peroxidases. 

Oxidation of organic contaminants by microorganisms is one of the basic metabolic reactions in the subsurface and involves the presence of a group of oxidative enzymes such as peroxidases, lactases, and mixed-function oxidizes. Major oxidative reactions that may occur in the subsurface are presented and explained in Table 15.2. 

Compound I is a two-electron oxidized enzyme intermediate containing a oxyferryl iron and a porphyrin cation radical while compound II is an one-electron oxidized intermediate (13), With lignin pa oxidase, as with other peroxidases, the substrate oxidation products are fir radicals which undergo nonenzymatic disproportionation reactions to give rise to the final products. 

Oxidizing enzymes have also been used as key catalysts in mulH-step reacHons for the production of antibacterial and anHviral agents. Horseradish peroxidase and chloroperoxidase have been used in the producHon of the macrocycHc glyco-... 

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

Lignin peroxidase activity, (i.e., peroxide-dependent oxidation of veratryl alcohol at pH 3) was not detected over the 30 days tested, while laccase appeared at day 7. Culture medium from day 7 onwards could also oxidize veratryl alcohol to aldehyde with concomitant conversion of oxygen to hydrogen peroxide. This activity, which was optimal at pH 5.0, was named veratryl alcohol oxidase (VAO). The extracellular oxidative enzyme activities (laccase and veratryl alcohol oxidase) could be separated by ion-exchange chromatography (Figure 2). Further chromatography of the coincident laccase and veratryl alcohol oxidase (peak 2), as described elsewhere (25) resulted in the separation of two veratryl alcohol oxidases from the laccase. 

The products of oxidation are easily visualized shortly after ozone treatments but no claim should be made that phenolics or the oxidative enzymes, polyphenol oxidase, phenolase or peroxidase... 

Major enzymes catalyzing flavonoid reactions are oxidative enzymes (i.e., polyphenoloxi-dases and peroxidases) arising from grape but also from molds contaminating them. Various hydrolytic enzymes (glycosidases, esterases), excreted by the fermentation yeasts or fungi or present in preparations added for technological purposes (e.g., pectinases), are also encountered in wine. 

A typical chemical system is the oxidative decarboxylation of malonic acid catalyzed by cerium ions and bromine, the so-called Zhabotinsky reaction this reaction in a given domain leads to the evolution of sustained oscillations and chemical waves. Furthermore, these states have been observed in a number of enzyme systems. The simplest case is the reaction catalyzed by the enzyme peroxidase. The reaction kinetics display either steady states, bistability, or oscillations. A more complex system is the ubiquitous process of glycolysis catalyzed by a sequence of coordinated enzyme reactions. In a given domain the process readily exhibits continuous oscillations of chemical concentrations and fluxes, which can be recorded by spectroscopic and electrometric techniques. The source of the periodicity is the enzyme phosphofructokinase, which catalyzes the phosphorylation of fructose-6-phosphate by ATP, resulting in the formation of fructose-1,6 biphosphate and ADP. The overall activity of the octameric enzyme is described by an allosteric model with fructose-6-phosphate, ATP, and AMP as controlling ligands. 

Walker, G. A. and Kilgour, G. L. 1965. Pyridine nucleotide oxidizing enzymes of Lactobacillus casei. II. Oxidase and peroxidase. Arch. Biochem. Biophys. Ill, 534-539. 

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