Kinetics peroxidase reactions

Similar experiments were performed in different dioxane concentrations ranging from 60% to 100% v/v. No reaction was observed in pure dioxane or in dioxane supplemented with 1% aqueous buffer. All peroxidase reactions in dioxane concentrations below 95% v/v obeyed Michaelis-Menten kinetics (Fig. 3). The values of apparent Km and kcat are plotted in Figures 4 and 5, respectively. The maximum value of Km at 80% dioxane remains poorly understood. It is possible that the enzyme undergoes a conformational change in dioxane concentrations above 80% which enhances the binding of the p-cresol (and hence a drop in apparent values of Km above this concentration of dioxane). The significant drop in kcat above 80% dioxane is consistent with this speculation as conformational changes in the peroxidase would almost surely lead to diminished catalytic activity. 

The formation of a reversible Michaelis-Menten-type complex of the enzyme and ferrocytochrome c [ES1S2 in Eq. (3) ] can be postulated from initial steady-state kinetics of the cytochrome c peroxidase reaction (17). Since cytochrome c peroxidase and cytochrome c are acidic and basic proteins, respectively, their interaction may be governed principally by electrostatic attraction. This assumption is further supported by the fact that several polycations which reversibly and irreversibly bind cytochrome c peroxidase inhibit its enzymic activity in competition with ferrocytochrome c 17,62). 

The peroxidase reaction (Figure 12.7a) can function in the absence of cyclooxygenase activity, because the intermediate product (prostaglandin G2) can be provided by another enzyme molecule. In accord with this model, a sample of cyclooxygenase, when expressed recombinantly and in the absence of arachidonic acid substrate, will initially be inactive, but it will exhibit burst kinetics upon first contact with arachidonic acid, due to the cascading activation of more and more enzyme molecules by PGG2". ... 

H Wariishi, HB Dunford, ID MacDonald, MH Gold. Manganese peroxidase from the hgnin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism. J Biol Chem 246(6) 3335-3340, 1989. 

They confirmed that conversions from I to II and from II to free peroxidase correspond to single-equivalent reductions. Since II was observable as an intermediate in the steady state of the ordinary peroxidase reactions, it was suggested that organic molecules are oxidized to semi-oxidized free radicals. We have confirmed the mechanism by titrating the radicals with suitable electron acceptors (26) and finally by using ESR spectroscopy (29). Kinetic analysis of free radical formation during the peroxidase reaction made it possible to conclude that most of the free radicals formed in Reactions 9 and 10 dismutate with each other rather than react further with the enzyme in the oxidized state (30). 

KINETIC AND ISOTROPIC STUDIES OF ACID-BASE CATALYSIS IN PEROXIDASE REACTIONS... 

That these mechanisms are valid has been concluavely shown by Chance (1-4), who studied the kinetics intensively by means of rapid optical methods that allow him to measure the rates of the separate reaction steps. He could show (4,101) that k[, the reaction constant for over-all catalase activity, is related to ki and k i of equations (4) and (5). But ki and 4 cannot be determined individually from the over-all reaction kinetics unless the ratio of the steady state concentrations of the enzyme-substrate compound to the free enzyme is known. In the case of peroxidatic activities where the substrate and donor are different molecules, their relative concentrations determine whether h or h is measured by the over-all activity usually an ill-de6ned mixture of the two reaction velocity constants is measured. The conditions appropriate for the evaluation of ki and kt from the over-all activity are discussed in a recent paper (4) by Chance, and the calculation of ki from the kinetics of peroxidase reactions and the standard peroxidase activity unit PZ (see p. 389) has also been carried out. Any sound procedure for activity determination should depend to as great an extent as possible upon the measurement of only one reaction velocity constant of the enzymic mechanism. 

Baek, H.K. and van Wart, H.E., Elementary steps in the reaction of horseradish peroxidase with several peroxides kinetics and thermodynamics of formation of compound 0 and compound I, J. Am. Chem. Soc., 114, 718-725, 1992. 

This approach uses a kinetic sequential principle to carry out multicomponent CL-based determinations. In fact, when the half-lives of the CL reactions involved in the determination of the analytes in mixture are appreciably different, the CL intensity-versus-time curve exhibits two peaks that are separate in time (in the case of a binary mixture) this allows both analytes to be directly determined from their corresponding calibration plots. In general, commercially available chemiluminometers have been used in these determinations, so the CL reaction was initially started by addition of one or two reaction ingredients. Thus, in the analysis of binary mixtures of cysteine and gluthatione, appropriate time-resolved response curves were obtained provided that equal volumes of peroxidase and luminol were mixed and saturated with oxygen and that copper(H) and aminothiol solutions were simultaneously injected [62, 63],... 

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 ... 

The structure of HRP-I has been identified as an Fe(IV) porphyrin -ir-cation radical by a variety of spectroscopic methods (71-74). The oxidized forms of HRP present differences in their visible absorption spectra (75-77). These distinct spectral characteristics of HRP have made this a very useful redox protein for studying one-electron transfers in alkaloid reactions. An example is illustrated in Fig. 2 where the one-electron oxidation of vindoline is followed by observing the oxidation of native HRP (curve A) with equimolar H202 to HRP-compound I (curve B). Addition of vindoline to the reaction mixture yields the absorption spectrum of HRP-compound II (curve C) (78). This methodology can yield useful information on the stoichiometry and kinetics of electron transfer from an alkaloid substrate to HRP. Several excellent reviews on the properties, mechanism, and oxidation states of peroxidases have been published (79-81). 

Cytochrome c and cytochrome c peroxidase (ccp) are physiological partners in the ccp reaction cycle structural, thermodynamic, and kinetic data are available for the protein-protein interaction [69-72]. A model indicates that the cyt c/ccp complex is stabilized by specific salt bridges with the hemes in parallel planes the Fe-Fe distance is 24 A, and the edge-edge distance is 16 A [70]. 

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). 

The catalase-peroxidases present other challenges. More than 20 sequences are available, and interest in the enzyme arising from its involvement in the process of antihiotic sensitivity in tuherculosis-causing bacteria has resulted in a considerable body of kinetic and physiological information. Unfortunately, the determination of crystallization conditions and crystals remain an elusive goal, precluding the determination of a crystal structure. Furthermore, the presence of two possible reaction pathways, peroxidatic and catalatic, has complicated a definition of the reaction mechanisms and the identity of catalytic intermediates. There is work here to occupy biochemists for many more years. 

Effect of pH on Lignin Peroxidase Catalysis. The oxidation of organic substrates by lignin peroxidase (Vmax) has a pH optimum equal to or possibly below 2. Detailed studies have been performed on the pH dependency of many of the individual reactions involved in catalysis. The effect of pH on the reaction rates between the isolated ferric enzyme, compounds I or II and their respective substrates has been studied. Rapid kinetic data indicate that compound I formation from ferric enzyme and H2O2 is not pH dependent from pH 2.5-7.5 (75,16). Similar results are obtained with Mn-dependent peroxidase (14). This is in contrast to other peroxidases where the pKa values for the reaction of ferric enzyme with H2O2 are usudly in the range of 3 to 6 (72). 

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),... 

An efficient method to prepare enantiomerically enriched hydroperoxides is the enzymatic kinetic resolution of racemic hydroperoxides using different kinds of enzymes (mainly lipases, chloroperoxidase, horseradish peroxidase). However, the scope of these reactions may be limit by the narrow substrate specificity of the enzyme. 

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. 

This problem was solved by Adam and coworkers in 1994-1998. They presented a high-yielding and diastereoselective method for the preparation of epoxydiols starting from enantiomerically pure allyhc alcohols 39 (Scheme 69). Photooxygenation of the latter produces unsaturated a-hydroxyhydroperoxides 146 via Schenck ene reaction. In this reaction the (Z)-allylic alcohols afford the (5, 5 )-hydroperoxy alcohols 146 as the main diastereomer in a high threo selectivity (dr >92 8) as racemic mixmre. The ( )-allylic alcohols react totally unselectively threolerythro 1/1). Subsequent enzymatic kinetic resolution of rac-146 threolerythro mixture) with horseradish peroxidase (HRP) led to optically active hydroperoxy alcohols S,S) and (//,5 )-146 ee >99%) and the... 

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... 

Horseradish peroxidase catalysed kinetic resolution of racemic secondary hydroperoxides has been described by Adam et al. [79]. The reaction yields (i )-hy-droperoxides up to ee>99% and (S)-alcohols up to ee>97%. Optically active hydroperoxides as potential stereoselective oxidants can be obtained by this process. 

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. 

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