Peroxidase enzyme intermediate

In order for the cyclooxygenase to function, a source of hydroperoxide (R—O—O—H) appears to be required. The hydroperoxide oxidizes a heme prosthetic group at the peroxidase active site of PGH synthase. This in turn leads to the oxidation of a tyrosine residue producing a tyrosine radical which is apparendy involved in the abstraction of the 13-pro-(5)-hydrogen of AA (25). The cyclooxygenase is inactivated during catalysis by the nonproductive breakdown of an active enzyme intermediate. This suicide inactivation occurs, on average, every 1400 catalytic turnovers. 

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. 

Since compound II has a Soret peak at 420 nm and the oxycomplex 416 nm, the equilibrated solution of compound II, H2O2, and compound III should exhibit an intermediate absorption peak. The existence of such an equilibrium has been established with HRP (22) and was also observed with lignin peroxidase (Cai, D., and Tien, M., unpublished results 23). Wariishi and Gold (25) recently proposed the existence of a new enzyme intermediate when compound in is formed in the presence of H2O2. They referred to this intermediate as compound III and suggested it to be a complex between compound III and H2O2. The existence of such an intermediate was based primarily on the Soret spectral properties where a 419 nm maximum was observed. In contrast, we see no evidence for existence of such a complex. The observed 419 nm absorbance maximun can be best explained by the existence of both compounds II and III in solution. 

The tyramide amplification technique is based on the ability of phenolic compounds to become oxidized to highly reactive and unstable intermediates (8). When biotinyl tyramide is oxidized, dimerization with electron-rich aromatic compounds, such as those found in protein molecules, occurs (9). This reaction can be harnessed in immunohistochemistry to generate highly reactive biotinyl-tyramide intermediates that bind rapidly to protein molecules in the immediate vicinity of peroxidase enzymes. This reaction results in the deposition... 

Fig. 2. Mechanism of haem peroxidases and catalases. Main reaction pathways and oxidation state of iron in haem peroxidases and catalases. Enzymes illustrated LPO, lactoperoxidase MPO, myeloperoxidase TPO, thyroid peroxidase Cat., catalase CCP, cytochrome c peroxidase HRP, horseradish peroxidase. Fe(III), Ferric (met) form of enzyme Compounds I, II and III enzyme intermediates.

The function of peroxidase enzymes is the activation of HOOH to provide two oxidizing equivalents for the oxidation of a variety of substrates. The interaction of horseradish peroxidase (HRP, an iron(in) heme that has a proximal imidazole) with HOOH results in the formation of a green reactive intermediate known as Compound I. It is reduced by one electron to give a red reactive intermediate, Compound II. Both of these intermediates contain a single oxygen atom from HOOH, and Compound I is two oxidizing equivalents above the iron(III)-heme state with a magnetic moment equivalent to three unpaired electrons (S = 3/2). A recent extended X-ray-absorption fine-structure (EXAFS) study summarizes the physical data in support of (por+-)Fe =0 as a... 

In the course of the reaction a number of substrates, termed redogenic , were capable of reducing a number of electron acceptors, such as oxygen, methylene blue, and cytochrome c, and the reducing agent was the free radical intermediate. This same peroxidase system was found to be capable of generating a free radical from chlorpromazine [66], which was capable of reducing the enzyme intermediate peroxidase compound II to peroxidase a result not obtained with the other substrates. 

Peroxidases are known to catalyze the one electron oxidation of a wide range of structurally diverse organic and inorganic compounds, Fig. (1). The catalytic cycle, first described by Chance [26] for horseradish peroxidase, may be described as follows. H2O2 oxidizes the ferric form of the enzyme (Felll), in a two electronic oxidation, to yield the enzyme intermediate compound I (Col) ... 

As one may expect from the peroxidase reaction mechanism described in Eqs. (2-4), the reactivity of the enzyme intermediates towards a particular substrate may be estimated a priori on the basis of the thermodynamic driving force of these two electron-transfer reactions, which is directly related with the difference between the oxidation/reduction potentials of both the enzyme active intermediates (i.e.. Col and Coll) and the substrate radicals. Thus, the thermodynamic driving force for the reaction of Col (or Coll) with the reducing substrates is the difference between the mid-point potentials of the CoI/CoII (or CoII/Felll) and the substrate radical/substrate (R, Hr/RH) redox couples ... 

Mabronk, P. A., The use of nonaqueous media to probe biochemically significant enzyme intermediates the generation and stabilization of horseradish peroxidase componnd II in neat benzene solntion at room temperatnre, J. Am. Chem. Soc., 117, 2141-2146, 1995. 

The first step in peroxidase catalysis is the formation of a two-electron oxidized enzyme intermediate on reaction of peroxide (ROOH, R = H, alkyl, or aryl) with the resting ferric form of the enzyme. This intermediate, which stores the two oxidizing equivalents of the peroxide, is termed compound I. An Fe -OOH intermediate, or ES complex. 

Hll. Hodgson, M., and Jones, P., Enhanced chemiluminescence in the peroxidase-luminol-hydro-gen peroxide system Anomalous reactivity of enhancer phenols with enzyme intermediates. J. Biolumin. Chemilumin. 3, 21-25 (1989). 

Ferryl complexes have been implicated in the reaction mechanisms of peroxidases and cytochromes P450. 38,1596 Pqj. horseradish peroxidase, two intermediates are spectroscopically detectable. Compound I, formed upon addition of peroxide to the resting Fe form of the enzyme, is a green species that is formally two oxidation levels higher than the resting state, and is widely believed to consist of an (Fe =0) + unit complexed by a porphyrin jt-cation radical. The [(P" ) Fe =0]+ complexes are discussed in Section 9. Compound II, which is red, and is obtained upon one-electron reduction of Compound I, also possesses a (Fe =0) + unit, in this case complexed by a normal porphyrin dianion, PFe =0. The fifth ligand, provided by the protein in the various enzymes, is a cysteine thiolate for the cytochromes nitric oxide synthases... 

An unusual kinetic feature of COX is the autoinactivation (suicide inactivation) of the enzyme both the cyclooxygenase and peroxidase activities are inactivated during catalysis as the result of non-productive breakdown of active enzyme intermediates. The chemical changes in the protein that accompany this process are unknown, and although the biological relevance of this inactivation is unclear, it may constitute a cmde regulatory mechanism of cellular prostaglandin biosynthesis (Smith et al, 2000). 

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