Peroxidase-catalyzed reactions kinetic

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. 

Peroxidases (EC 1.11.1.7) catalyze the reduction of hydrogen peroxide or alkyl hydroperoxides while a wide range of substrates act as electron donors. The mechanism of peroxidase catalyzed reactions has been intensively studied (see reviews [201,207-210]). The kinetics of catalysis reveals a ping-pong mechanism. In the first step, the peroxide binds to a free coordination site of iron (Fe ) and is reduced to water (or an alcohol ROH) in a rapid two-electron process, whereby compound I is formed as the stable primary intermediate ... 

As shown in Table 12,H202 and fBuOOH have been used frequently as oxygen donors in peroxidase-catalyzed sulfoxidations. Other achiral oxidants, e.g. iodo-sobenzene and peracids, are not accepted by enzymes and, therefore, only racemic sulfoxides were found (c.f. entries 34-36). Interestingly, racemic hydroperoxides oxidize sulfides to sulfoxides enantioselectively under CPO catalysis [68]. In this reaction, not only the sulfoxides but also the hydroperoxide and the corresponding alcohol were produced in optically active form by enzyme-catalyzed kinetic resolution (cf. Eq. 3 and Table 3 in Sect. 3.1). 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Using a similar approach to that used in the derivation of the Michaelis-Menten model, the kinetic equations of the peroxidase-catalyzed removal of an aromatic substrate were derived from the reaction pathways illustrated in Fig. 2 [92]. The rate of change of the aromatic compound concentration can be written as... 

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

The absolute amounts of aniline covalently bonded to the soil fulvic acid in the presence and absence of the peroxidase were not measured. However, the relative signal to noise ratios obtained in the NMR spectra indicate that significantly more aniline was taken up by the fulvic acid in the enzyme catalyzed reaction. It should also be pointed out that, in the execution of the peroxidase experiment, the solution containing the fulvic acid, aniline, and peroxidase darkened instantaneously upon addition of the hydrogen peroxide, indicating significantly faster kinetics than in the nonenzyme reaction. 

Oxidation of lAA catalyzed by peroxidase and lAA oxidase with accompanying decarboxylation has been extensively studied [ 17,18,20,21 ] and enzymatic kinetics of horseradish peroxidase and reaction products of lAA have been elucidated [8,9, 12, 14]. In some plant species, however, peroxidative decarboxylation of lAA was reported to be a minor component in Zea seedlings [6, 15] and in Pinus seeds [7], where IA A was oxidized to Oxl A A [ 19]. In the present report, we show another lAA oxidation system without decarboxylation in Vicia seedlings, where lAA-Asp was oxidized to dioxindole-3-acetic acid (DIA) conjugates, and a possibility that peroxidase could contribute to the lAA-Asp oxidation was also examined. 

The enzymatic reaction kinetics on the HRP-catalyzed oxidation of p-cresol in aqueous 1,4-dioxane or methanol showed that the cataljdic turnover niunber and Michaelis constant were larger than those in water (235). Numerical and Monte Carlo simulations of the peroxidase-catalyzed polymerization of phenols were demonstrated (236). The simulations predicted the monomer reactivity and polymer molecular weight, leading to synthesis of polymers with specific molecular weight and index. In an aqueous 1,4-dioxane, the formation of monomer aggregate was observed (237), which might elucidate the specific polymerization behaviors in such a medium. 

The half-order kinetics indicate that the role played by cyanide is a complex one. But the results support the view that the stimulation by cyanide is due to an effect on the reaction steps involving oxygen, which Eq. (11) suggests may be similar in the peroxidase catalyzed and nonenzymic oxidations. The inhibition at higher DHF concentrations would then be due to the complex-forming reaction between cyanide and peroxidase as, in the presence of more DHF, much more free ferric peroxidase will be present in the reaction steady state. 

A number of autoxidation reactions exhibit exotic kinetic phenomena under specific experimental conditions. One of the most widely studied systems is the peroxidase-oxidase (PO) oscillator which is the only enzyme reaction showing oscillation in vitro in homogeneous stirred solution. The net reaction is the oxidation of nicotinamide adenine dinucleotide (NADH), a biologically vital coenzyme, by dioxygen in a horseradish peroxidase enzyme (HRP) catalyzed process ... 

Studies on the effect of pH on peroxidase catalysis, or the heme-linked ionization, have provided much information on peroxidase catalysis and the active site structure. Heme-linked ionization has been observed in kinetic, electrochemical, absorption spectroscopic, proton balance, and Raman spectroscopic studies. Kinetic studies show that compound I formation is base-catalyzed (72). The pKa values are in the range of 3 to 6. The reactions of compounds I and II with substrates are also pH-dependent with pKa values in a similar range (72). Ligand binding (e.g. CO, O2 or halide ions) to ferrous and ferric peroxidases is also pH-dependent. A wide range of pKa values has been reported (72). The redox potentials of Fe3+/Fe2+ couples for peroxidases measured so far are all affected by pH. The pKa values are between 6 and 8, indicative of an imidazole group of a histidine residue (6, 31-33),... 

Hoft reported about the kinetic resolution of THPO (16b) by acylation catalyzed by different lipases (equation 12) °. Using lipases from Pseudomonas fluorescens, only low ee values were obtained even at high conversions of the hydroperoxide (best result after 96 hours with lipase PS conversion of 83% and ee of 37%). Better results were achieved by the same authors using pancreatin as a catalyst. With this lipase an ee of 96% could be obtained but only at high conversions (85%), so that the enantiomerically enriched (5 )-16b was isolated in poor yields (<20%). Unfortunately, this procedure was limited to secondary hydroperoxides. With tertiary 1-methyl-1-phenylpropyl hydroperoxide (17a) or 1-cyclohexyl-1-phenylethyl hydroperoxide (17b) no reaction was observed. The kinetic resolution of racemic hydroperoxides can also be achieved by chloroperoxidase (CPO) or Coprinus peroxidase (CiP) catalyzed enantioselective sulfoxidation of prochiral sulfides 22 with a racemic mixmre of chiral hydroperoxides. In 1992, Wong and coworkers and later Hoft and coworkers in 1995 ° investigated the CPO-catalyzed sulfoxidation with several chiral racemic hydroperoxides while the CiP-catalyzed kinetic resolution of phenylethyl hydroperoxide 16a was reported by Adam and coworkers (equation 13). The results are summarized in Table 4. 

As already reported in Section II.A.2, the enzymes chloroperoxidase (CPO) and Copri-nus peroxidase (CiP) catalyze the enantioselective oxidation of aryl alkyl sulfides. If a racemic mixture of a chiral secondary hydroperoxide is used as oxidant, kinetic resolution takes place and enantiomerically enriched hydroperoxides and the corresponding alcohols can be obtained together with the enantiomerically enriched sulfoxides. An overview of the results obtained in this reaction published by Wong and coworkers, Hoft and... 

Synthetic iron porphyrin complexes such as Fe(TPP) (tetraphenylporphyrin = TPP), Fe(TMP) (Tetramesitylporphyrin = TMP), and Fe(TDCPP) (tetrakis (dichlorophenyl)porphyrin = TDCPP) (Fig. 9) have been used as models for P450 and peroxidase (9, 50-54). Early pioneering work showed that epoxida-tion catalyzed by Feln(TPP) was successfully carried out by the use of iodosylbenzene (Ph—1=0) as an oxidant (50). A very interesting feature of this model epoxidation is that the cis olefin is readily oxidized while the trans olefin is hardly oxidized (e.g., d.v-stylbene can be oxidized in 80% yield, but fraws-stylbene gave only a trace amount of the epoxide under the same conditions) (50, 55). Most of the model reactions are carried out in homogeneous organic solvents such as chloroform, dichloromethane, and acetonitrile, thus, the c/.v-epoxidation is expected to be a kinetically favorable process over the trans-epoxidation. 

LiP catalyzes the oxidation of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) to veratryl aldehyde. Since this reaction can be easily followed at 310 nm, it is the basis for the standard assay for this enzyme (26,27). The enzyme exhibits normal saturation kinetics for both veratryl alcohol and H202 (28,43). Steady-state kinetic results Indicate a ping-pong mechanism in which H202 first oxidizes the enzyme and the oxidized intermediate reacts with veratryl alcohol (43). The enzyme has an extremely low pH optimum ( 2.5) for a peroxidase (43,44) however, the rate of formation of compound I (kx, Fig. 2) exhibits no pH dependence from 3.0-7.0 (45,46). Addition of excess veratryl alcohol at pH 3.0 results in the rapid conversion of... 

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