Peroxidase rate constant

Enzyme-substrate complex Compound III Elorseradish peroxidase Forward rate constant, L/mol-s Reverse rate constant, s 1 Rate constant, s Forward rate constant, L/mol-s Reverse rate constant, s 1 Rate constant, s Forward rate constant, L/mol-s Reverse rate constant, s 1 Rate constant, s Rate constant, s Inactivation constant, mM Constant, mM Constant, mM Pentachlorophenol... 

The electrochemical rate constants for hydrogen peroxide reduction have been found to be dependent on the amount of Prussian blue deposited, confirming that H202 penetrates the films, and the inner layers of the polycrystal take part in the catalysis. For 4-6 nmol cm 2 of Prussian blue the electrochemical rate constant exceeds 0.01cm s-1 [12], which corresponds to the bi-molecular rate constant of kcat = 3 X 103 L mol 1s 1 [114], The rate constant of hydrogen peroxide reduction by ferrocyanide catalyzed by enzyme peroxidase was 2 X 104 L mol 1 s 1 [116]. Thus, the activity of the natural enzyme peroxidase is of a similar order of magnitude as the catalytic activity of our Prussian blue-based electrocatalyst. Due to the high catalytic activity and selectivity, which are comparable with biocatalysis, we were able to denote the specially deposited Prussian blue as an artificial peroxidase [114, 117]. 

Figure 4.3. The catalytic cycle of horseradish peroxidase with ferulate as reducing substrate. The rate constants Ki, K2, and K3 represent the rate of compound I formation, rate of compound I reduction, and rate of compound II reduction, respectively.

Scheme 6. General mechanistic picture of catalysis by Fem-TAML activators of peroxides that underscores the peroxidase-like (peroxida-tic) and catalase-like (catalatic) activities of the catalysts. Here, S(red) is an oxidizable target substrate. All rate constants are conditional (pH dependent).

Rate Constants kV2 foe the Formation of Compound I from H202 and Selected Peroxidases or Catalases and for Non-Fenton Peroxidase- or Catalase-Related Activation of H202 Such as k by the Most Reactive Low-Molecular Weight Ieon(III) Complexes in Water... 

Entry Enzyme or metal complex with catalase- or peroxidase-like activity pH (T/°C) Rate constant/ M s 1 Refs. 

The rate constants k s are again pH dependent and the pH profiles for reactions in Eqs. (14) (Fig. 15) and (17), both catalyzed by 11, are similar. Eq. (16) has therefore also been applied for fitting the data for reaction in Eq. (17). The best-fit rate and equilibrium constants are summarized in Table V. For the reasons discussed above, the rate constants k3 (obtained by setting k2 = 0) are not included in Table V. The rate constants in Tables III and V indicate that the catalase-like rate constants are somewhat lower than the peroxidase-like rate constants. 

The molecular masses of heme catalases are usually significantly higher as compared with peroxidases. If expressed in Lg-1s-1, rate constants for the Fem-TAML activators when compared with catalase from beef liver, which has a molecular weight 250,000 gmol-1 (Table IV, entry 13) (89), look very impressive, viz. 17 L g 1 s-1 for 11 vs. 22 L g 1 s 1 for the enzyme. Nevertheless, the catalase-like activity of the Fem-TAML activators can be suppressed by the addition of electron donors -it is negligible in the presence of the substrates tested in this work. In Nature, catalases display only minor peroxidase-like activity (79) because electron donors bulkier than H202 cannot access the deeply buried active sites of these massive enzymes (90). The comparatively unprotected Fem-TAML active sites are directly exposed to electron donors such that the overall behavior is determined by the inherent relative reactivity of the substrates. 

The rate constants of kn and kx obtained using Eq. (24) reveal that (i) the activity of Fem-TAMLs in bleaching Safranine O (k ) increases more than 10-fold when the tail ethyl groups of la are replaced by fluorine atoms in lk. The rate constant kn for lk equals l(rM 1s 1 at 25°C, a value that corresponds to those found for the reactivity of horseradish peroxidase Compound II... 

For a long time one question remained unanswered the efficiency of the Fenton reaction as the in vivo producer of hydroxyl radicals due to the low rate of Reaction (2) (the rate constant is equal to 42.11 mol 1 s 1 [18]). It is known that under in vitro conditions the rate of Fenton reaction can be sharply enhanced by chelators such as EDTA, but for a long time no effective in vivo chelators have been found. From this point of view new findings obtained by Chen and Schopfer [19] who found that peroxidases catalyze hydroxyl radical formation in plants deserve consideration. These authors showed that horseradish peroxidase (HRP) compound III is a catalyst of the Fenton reaction and that this compound is one to two orders of magnitude more active than Fe EDTA. 

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. 

Various hydroxyl and amino derivatives of aromatic compounds are oxidized by peroxidases in the presence of hydrogen peroxide, yielding neutral or cation free radicals. Thus the phenacetin metabolites p-phenetidine (4-ethoxyaniline) and acetaminophen (TV-acetyl-p-aminophenol) were oxidized by LPO or HRP into the 4-ethoxyaniline cation radical and neutral V-acetyl-4-aminophenoxyl radical, respectively [198,199]. In both cases free radicals were detected by using fast-flow ESR spectroscopy. Catechols, Dopa methyl ester (dihydrox-yphenylalanine methyl ester), and 6-hydroxy-Dopa (trihydroxyphenylalanine) were oxidized by LPO mainly to o-semiquinone free radicals [200]. Another catechol derivative adrenaline (epinephrine) was oxidized into adrenochrome in the reaction catalyzed by HRP [201], This reaction can proceed in the absence of hydrogen peroxide and accompanied by oxygen consumption. It was proposed that the oxidation of adrenaline was mediated by superoxide. HRP and LPO catalyzed the oxidation of Trolox C (an analog of a-tocopherol) into phenoxyl radical [202]. The formation of phenoxyl radicals was monitored by ESR spectroscopy, and the rate constants for the reaction of Compounds II with Trolox C were determined (Table 22.1). 

The rate constants for two-electron reaction (8) (A+) and one-electron reaction (19) (k 19) are cited in Table 22.3. As seen from Table 22.3, ks values are about 100 1000 times greater than kig values therefore, the production of superoxide (or hydroperoxyl radical) by peroxidases might play an insignificant role compared to the two-electron oxidation of hydroperoxides. 

At the same time the interaction of superoxide with MPO may affect a total superoxide production by phagocytes. Thus, the superoxide adduct of MPO (Compound III) is probably quantitatively formed in PMA-stimulated human neutrophils [223]. Edwards and Swan [224] proposed that superoxide production regulate the respiratory burst of stimulated human neutrophils. It has also been suggested that the interaction of superoxide with HRP, MPO, and LPO resulted in the formation of Compound III by a two-step reaction [225]. Superoxide is able to react relatively rapidly with peroxidases and their catalytic intermediates. For example, the rate constant for reaction of superoxide with Fe(III)MPO is equal to 1.1-2.1 x 1061 mol 1 s 1 [226], and the rate constants for the reactions of Oi and HOO with HRP Compound I are equal to 1.6 x 106 and 2.2 x 1081 mol-1 s-1, respectively [227]. Thus, peroxidases may change their functions, from acting as prooxidant enzymes and the catalysts of free radical processes, and acquire antioxidant catalase properties as shown for HRP [228] and MPO [229]. In this case catalase activity depends on the two-electron oxidation of hydrogen peroxide by Compound I. 

The most important physiological nitrogen substrate of peroxidases is undoubtedly nitric oxide. In 1996, Ishiropoulos et al. [252] suggested that nitric oxide is able to interact with HRP Compounds I and II. Glover et al. [253] measured the rate constants for the reactions of NO with HRP Compounds I and II (Table 22.2) and proposed that these reactions may occur in in vivo inflammatory processes. The interaction of NO with peroxidases may proceed by two ways through the NO one-electron oxidation or the formation of peroxidase NO complexes. One-electron oxidation of nitric oxide will yield nitrosonium cation NO+ [253,254], which is extremely unstable and rapidly hydrolyzed to nitrite. On the other hand, in the presence of high concentrations of nitric oxide and the competitor ligand Cl, the formation of peroxidase NO complexes becomes more favorable. It has been shown [255]... 

Nitric oxide and nitrite react with other peroxidase enzymes such as horseradish peroxidase (HRP) (138a,139), lactoperoxidase (138a) and eosinophil peroxidase (140) similarly. The rate constants for reaction of NO with compounds I and II in HRP were found to be 7.0 x 105M 1s 1 and 1.3 x 106M 1s 1, respectively (139). Catalytic consumption of NO as measured by an NO sensitive electrode in the presence of HRP compounds I and II is shown in Fig. 5 where accelerated consumption of NO is achieved even in deoxygenated solutions (140). 

The enzyme horseradish peroxidase is a hemoprotein and the region of the Soret band exhibits large differences between the position and extinction coefficients of the uncombined and combined forms. Both forms were first studied by spectrophotometry, but the E—S complexes were 0 labile that they could not be examined extensively by any other spectroscopic method. Using rapid-scanning spectrophotometry and rapid mixing, Chance was able to distinguish the spectra of compound I and II and determine the various rate constants of the multistep reaction with rather poor precision. 

Although compound I formation is not influenced by pH, reactions of compounds I and II are significantly affected by pH. These reactions are acid-catalyzed 16,17). The rate constant for the oxidation of veratryl alcohol or fenocyanide by lignin peroxidase compound I is 10 times greater at pH 3.5 than at pH 6.0. The enhancement in rate is of the same magnitude for compound II reacting with veratryl alcohol. Therefore, the observed pH dependency for Vmax in catalysis is due to the pH-dependent reactions between the compounds I and n and the reducing substrates. 

Mathematical Treatment of the Results. In almost all experiments inactivation followed first order kinetics with a high correlation. The half-lives of lignin peroxidase were calculated from the following equation, where k is a rate constant of inactivation ... 

Similarly, this amphiphilic polymer micelle was also used to dismpt the complex between cytochrome c (Cc) and cytochrome c peroxidase (CcP Sandanaraj, Bayraktar et al. 2007). In this case, we found that the polymer modulates the redox properties of the protein upon binding. The polymer binding exposes the heme cofactor of the protein, which is buried in the protein and alters the coordination environment of the metal. The exposure of heme was confirmed by UV-vis, CD spectroscopy, fluorescence spectroscopy, and electrochemical kinetic smdies. The rate constant of electron transfer (fc°) increased by 3 orders of magnimde for the protein-polymer complex compared to protein alone. To establish that the polymer micelle is capable of disrupting the Cc-CcP complex, the polymer micelle was added to the preformed Cc-CcP complex. The observed for this complex was the same as that of the Cc-polymer complex, which confirms that the polymer micelle is indeed capable of disrupting the Cc-CcP complex. 

Cyclic voltammetry has been also used for estimation of the rate constants for oxidation of water-soluble ferrocenes in the presence of HRP (131). There is a perfect match between the data obtained spectrophotometrically and electrochemically (Table IV), which proves that the cyclic voltammetry reveals information on the oxidation of ferrocenes by Compound II. It is interesting to note that an enzyme similar to HRP, viz. cytochrome c peroxidase, which catalyzes the reduction of H202 to water using two equivalents of ferrocytochrome c (133-136), is ca. 100 times more reactive than HRP (131,137). The second-order rate constant equals 1.4 x 106 M-1 s 1 for HOOCFc at pH 6.5 (131). There is no such rate difference in oxidation of [Fe(CN)e]4- by cytochrome c peroxidase and HRP (8). These comparisons should not however create an impression that the enzymatic oxidation of ferrocenes is always fast. The active-R2 subunit of Escherichia coli ribonucleotide reductase, which has dinuclear nonheme iron center in the active site, oxidizes ferrocene carboxylic acid and other water-soluble ferrocenes with a rate constant of... 

Planar chirality is a valuable feature of the ferrocene chemistry (Chart 2B,C) (205). This unnatural chirality type has attracted attention of several groups. Sadeghi and co-workers have demonstrated that the planar chiral ferrocenes are recognized by cytochrome c peroxidase (206). The rate constants for the oxidation of R and S enantiomers by the wild type enzyme equal 2.9 x 106 and 1.6 x 106 M 1 s respectively. Interestingly, the enantioselectivity inverts for the aspartate 34 for lysine mutant and the rate constants become equal to 5.9 x 106 and 14.8 x 106 M-1 s-1, respectively. The discrimination of planar chiral ferrocenes is the case, but the stereoselectivity factors are lower than 3. 

Thus, the [Ru(phpy)(phen)2]+ ruthenacycle is a strikingly reactive electron donor for HRR High rate constants for other complexes are summarized in Table IX. Plant peroxidases from sources other than horseradish also show a high reactivity to cyclometalated ruthenium(II) complexes listed in Table IX (234). 

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