Kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase]

A kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase within the pH range of 3.7-9.0 has been carried out. It was shown that the reaction of o-dianisidine peroxidase oxidation obeys the Michaelis--Menten kinetics; the kcat and Km values within the pH range used were determined. The optimum of peroxidase catalytic activity during o-dianisidine oxidation was observed at pH 5.0-6.0. The kinetic pattern of the reaction is discussed. It was demonstrated that deprotonation of the group at pK 6.5 decreases the kcat value 60 times. At pH greater than 8.0 an additional ionogenic group controls the enzyme activity.     

Inhibition and inactivation of horseradish peroxidase by thiourea

The kinetics of horseradish peroxidase (EC 1.11.1.7)-catalyzed oxidation of o-dianisidine by hydrogen peroxide in the presence of thiourea were studied. At the first, fast step of this process thiourea acts as a competitive reversible inhibitor with respect to o-dianisidine (Ki = 0.22 mM). The formation of a thiourea-peroxidase complex was determined by the increase in the absorbance at A495 and A638 of the enzyme. The dissociation constant for the peroxidase-thiourea complex is equal to 2.0-2.7 mM. Thiourea is not a specific substrate of peroxidase during the oxidation reaction by H2O2, but is an oxidase substrate (although not a very active one) of peroxidase. The irreversible inactivation of the enzyme during its incubation with thiourea was studied. The first-order inactivation rate constant (kin) was shown to increase with a fall in the enzyme concentration. The curve of the dependence of kin on the initial concentration of thiourea shows a maximum at 5-7 mM. The enzyme inactivation is due to its modification by intermediate free radical products of thiourea oxidation. The inhibitors of the free radical reactions (o-dianisidine) protect the enzyme against inactivation. The degree of inactivation depends on concentrations and ratio of thiourea and peroxidase. A possible mechanism of peroxidase interaction with thiourea is discussed.     

Peroxidase activity of succinylated catalase

The catalase succinylation by succinic anhydride excess results in an almost complete dissociation of the enzyme into subunits possessing no catalase activity. The catalase subunits show the peroxidatic activity on o-dianisidine oxidation. The oxidation kinetics of this substrate by the succinylated enzyme was studied at various temperatures. The activation energy for this process is 10.1 kcal/mole. Within the temperature range of 31-65.5 degrees, the succinylated enzyme thermostability was studied by monitoring the peroxidatic activity decrease upon o-dianisidine oxidation. The activation energy for the succinylated catalase thermoinactivation equals to 15.5 kcal/mole. The peroxidatic activity of catalase subunits obtained by enzyme succinylation and acidic solution treatment was compared to that of horseradish peroxidase in the oxidation of the same substrate, i.e., o-dianisidine.     

Peroxidase of propionic acid bacteria

The peroxidase activity was found in Propionibacterium shermanii. Methods were developed to isolate and purify the enzyme. It was shown to be a heme-containing protein, specific to H2O2, stable at 20 to 30 degrees C and exerting the optimal action at pH 6.8 to 7.0. The rate of the enzyme-catalysed reaction was studied as a function of the enzyme and substrate concentrations. The Km was determined for H2O2 and o-dianisidine.     

Purification and characterization of a cationic peroxidase Cs in Raphanus sativus

A short distance migrating cationic peroxidase from Korean radish seeds (Raphanus sativus) was detected. Cationic peroxidase Cs was purified to apparent homogeneity and characterized. The molecular mass of the purified cationic peroxidase Cs was estimated to be about 44 kDa on SDS-PAGE. After reconstitution of apoperoxidase Cs with protohemin, the absorption spectra revealed a new peak in the Soret region around 400 nm, which is typical in a classical type III peroxidase family. The optimum pH of peroxidase activity for o-dianisidine oxidation was observed at pH 7.0. Kinetic studies revealed that the reconstituted cationic peroxidase Cs has Km values of 1.18 mM and of 1.27 mM for o-dianisidine and H2O2, respectively. The cationic peroxidase Cs showed the peroxidase activities for native substrates, such as coumaric acid, ferulic acid, and scopoletin. This result suggested that cationic peroxidase Cs plays an important role in plant cell wall formation during seed germination.     

Effect of strophanthin G on peroxidase oxidation kinetics of slowly oxidizable peroxidase substrates

Steady-state kinetics of thioproperazine, triftazine, aminazine, and o-dianisidine oxidation with hydrogen peroxide catalyzed by horseradish peroxidase were studied in the presence of strophanthin G. Values of the inhibition and activation constants (Ki, Ka) were determined in the pH range 5.0-7.5. At acidic pH, strophanthin G activated peroxidase during the oxidation of thioproperazine by the uncompetitive mechanism, and when triftazine was oxidized, the inhibition was noncompetitive. At pH > 6.0, the patterns of activation and inhibition changed to mixed-type during the peroxidase oxidation of thioproperazine and triftazine and to competitive inhibition of peroxidase with strophanthin G during the oxidation of aminazine. These effects are suggested to be due to an ionizable enzyme group of pK approximately 6.0. Strophanthin G inhibited free-radical oxidation of o-dianisidine via binding to the enzyme-substrate complex, preventing the generation of a stable semi-oxidized product of o-dianisidine, and thus inhibiting the enzyme by the anticompetitive mechanism. Mechanisms of oxidation of slowly and rapidly oxidizable substrates of peroxidase in the presence of strophanthin G are suggested.     

Detection and substrate selectivity of new microbial D-amino acid oxidases

In order to screen for new microbial D-amino acid oxidase activities a selective and sensitive peroxidase/o-dianisidine assay, detecting the formation of hydrogen peroxide was developed. Catalase, which coexists with oxidases in the peroxisomes or the microsomes and, which competes with peroxidase for hydrogen peroxide, was completely inhibited by o-dianisidine up to a catalase activity of 500 nkat ml(-)(1). Thus, using the peroxidase/o-dianisidine assay and employing crude extracts of microorganisms in a microplate reader, a detection sensitivity for oxidase activity of 0.6 nkat ml(-)(1) was obtained.Wild type colonies which were grown on a selective medium containing D-alanine as carbon, energy and nitrogen source were examined for D-amino acid oxidase activity by the peroxidase/o-dianisidine assay. The oxidase positive colonies possessing an apparent oxidase activity > 2 nkat g dry biomass(-)(1) were isolated. Among them three new D-amino acid oxidase-producers were found and identified as Fusarium oxysporum, Verticilium lutealbum and Candida parapsilosis. The best new D-amino oxidase producer was the fungus F. oxysporum with a D-amino acid oxidase activity of about 900 nkat g dry biomass(-)(1) or 21 nkat mg protein(-)(1). With regard to the use as a biocatalytic tool in biotechnology the substrate specificities of the three new D-amino acid oxidases were compared with those of the known D-amino acid oxidases from Trigonopsis variabilis, Rhodotorula gracilis and pig kidney under the same conditions. All six D-amino acid oxidases accepted the D-enantiomers of alanine, valine, leucine, proline, phenylalanine, serine and glutamine as substrates and, except for the D-amino acid oxidase from V. luteoalbum, D-tryptophane, D-tyrosine, D-arginine and D-histidine were accepted as well. The relative highest activities (>95%) were measured versus D-alanine (C. parapsilosis, F. oxysporum, T. variabilis), D-methionine (V. luteoalbum, R. gracilis), D-valine (T. variabilis, R. gracilis) and D-proline (pig kidney). The D-amino oxidases from F. oxysporum and V. luteoalbum were able to react with the industrially important substrate cephalosporin C although the D-amino acid oxidase from T. variabilis was at least about 20-fold more active with this substrate.As the results of our studies, a reliable oxidase assay was developed, allowing high throughput screening in a microplate reader. Furthermore, three new microbial D-amino acid oxidase-producers with interesting broad substrate specificities were introduced in the field of biotechnology.     

Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.     

Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane, a nonphenolic dimeric lignin model compound. The spectrum of the peroxidase showed a soret band at 405 nm, which disappeared after reduction with sodium dithionite, indicating that the enzyme is a hemoprotein. Testing the effects of various inhibitors on the enzyme activity showed that it is a bifunctional enzyme having catalase and peroxidase activities.     

Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane, a nonphenolic dimeric lignin model compound. The spectrum of the peroxidase showed a soret band at 405 nm, which disappeared after reduction with sodium dithionite, indicating that the enzyme is a hemoprotein. Testing the effects of various inhibitors on the enzyme activity showed that it is a bifunctional enzyme having catalase and peroxidase activities.     

Mechanism of combined oxidation of ascorbic acid and hydroquinone in the presence of horseradish peroxidase]

A steady-state kinetics of peroxidase cooxidation of ascorbic acid and hydroquinone catalyzed by horseradish peroxidase was studied. Ascorbic acid and hydroquinone were shown to be oxidized successively, and hydroquinone promoted the oxidation of ascorbic acid. Excess ascorbic acid inhibited peroxidase in the cooxidation of the substrates at pH 5-7. The values of catalytic constants, (kcat, K(m), and Ka) were determined. A possible activation mechanism of the peroxidation of ascorbic acid in the presence of hydroquinone was suggested, and its biological significance was considered.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Mechanistic features of lignin peroxidase-catalyzed oxidation of substituted phenols and 1,2-dimethoxyarenes

The steady state kinetic parameters Km and kcat for the oxidation of phenolic substrates by lignin peroxidase correlated with the presteady state kinetic parameters Kd and k for the reaction of the enzyme intermediate compound II with the substrates, indicating that the latter is the rate-limiting step in the catalytic cycle. ln Km and ln Kd values for phenolic substrates correlated with redox properties, unlike ln kcat and ln k. This finding suggests that in contrast to horseradish peroxidase, electron transfer is not the rate-limiting step during oxidation by lignin peroxidase compound II. A mechanism is proposed for lignin peroxidase compound II reactions consisting of an equilibrium electron transfer step followed by a subsequent rate-limiting step. Analysis of the correlation coefficients for linear relationships between ln Kd and ln Km and different calculated redox parameters supports a mechanism in which the acidic forms of phenols are oxidized by lignin peroxidase and electron transfer is coupled with proton transfer. 1,2-Dimethoxyarenes did not comply with the trend for phenolic substrates, which may be a result of more than one substrate binding site on lignin peroxidase and/or alternative binding modes. This behavior was supported by analogue studies with the 1,2-dimethoxyarenes veratric acid and veratryl aldehyde, both of which are not oxidized by lignin peroxidase. Inclusion of either had little effect on the rate of oxidation of phenolic substrates yet resulted in a decrease in the oxidation rate of 1,2-dimethoxyarene substrates, which was considerable for veratryl alcohol and less pronounced for 3,4-dimethoxyphenethylalcohol and 3,4-dimethoxycinnamic acid, in particular in the presence of veratric acid.     

Characterization of the hydroperoxide-reducing activity of human plasma

A peroxidase was identified in human plasma using a novel peroxidase assay. In this assay both the substrate 5-phenyl-4-pentenyl hydroperoxide (PPHP) and its reduction product, 5-phenyl-4-pentenyl alcohol (PPA) are quantitated by HPLC. Substrate specificity studies indicated that the peroxidase requires glutathione as reducing substrate. No reduction was detected using the classical heme peroxidase reducing substrates, phenol and hydroquinone. Peroxidase activity was not due to glutathione transferases. Failure to saturate the peroxidase activity with reduced glutathione and inhibition by Cd+2 indicated that it is probably selenium dependent. The enzyme appears to be different from erythrocyte glutathione peroxidase based on kinetic and immunological experiments. The apparent Km values for PPHP are 25 microM for erythrocyte peroxidase and 54 microM for plasma peroxidase at 0.5 mM reduced glutathione. Anti-peroxidase prepared against bovine erythrocyte glutathione peroxidase partially inhibited human erythrocyte peroxidase but did not inhibit human plasma peroxidase.     

Mechanisms of peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate

Peroxidase oxidation of o-dianisidine, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1-100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.     

Thyroid peroxidase selects the mechanism of either 1- or 2-electron oxidation of phenols, depending on their substituents

Unlike lactoperoxidase and horseradish peroxidase, thyroid peroxidase catalyzed the oxidation of hydroquinone mostly by way of 2-electron transfer. This conclusion could be derived from three independent experiments: ESR measurements of p-benzosemiquinone, trapping the unpaired electron by cytochrome c, and spectrophotometric analysis of catalytic intermediates of the enzymes. The 1-electron flux for hydroquinone oxidation was found to be 15-19% in the reaction of thyroid peroxidase, while it was nearly 100% in the reactions of lactoperoxidase and horseradish peroxidase. From the spectrophotometric analysis of the catalytic intermediates of enzyme, it was suggested that the mechanism of oxidation catalyzed by thyroid peroxidase changes from a 2-electron to a 1-electron type as the substituents at 2- and 6-positions of phenol become bulky or heavy. On the other hand, the mechanism was invariably a 1-electron type when the oxidation of phenols was catalyzed by lactoperoxidase or horseradish peroxidase. These three peroxidases all catalyzed 1-electron oxidation of ascorbate.     

Inhibition of horseradish peroxidase by N-ethylamide of o-sulfobenzoylacetic acid

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence of N-ethylamide of o-sulfobenzoylacetic acid (EASBA) at pH 5.0-7.5. In the oxidation of o-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases both K(m) and Vm in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.     

Metabolism of hydroquinone by human myeloperoxidase: mechanisms of stimulation by other phenolic compounds.

Hydroquinone, a metabolite of benzene, is converted by human myeloperoxidase to 1,4-benzoquinone, a highly toxic species. This conversion is stimulated by phenol, another metabolite of benzene. Here we report that peroxidase-dependent hydroquinone metabolism is also stimulated by catechol, resorcinol, o-cresol, m-cresol, p-cresol, guaiacol, histidine, and imidazole. In order to gain insights into the mechanisms of this stimulation, we have compared the kinetics of human myeloperoxidase-dependent phenol, hydroquinone, and catechol metabolism. The specificity (Vmax/Km) of hydroquinone for myeloperoxidase was found to be 5-fold greater than that of catechol and 16-fold greater than that of phenol. These specificities for myeloperoxidase-dependent metabolism inversely correlated with the respective one-electron oxidation potentials of hydroquinone, catechol, and phenol and suggested that phenol- and catechol-induced stimulation of myeloperoxidase-dependent hydroquinone metabolism cannot simply be explained by interaction of hydroquinone with stimulant-derived radicals. Phenol (100 microM), catechol (20 microM), and imidazole (50 mM) did, however, all increase the specificity (Vmax/Km) of hydroquinone for myeloperoxidase, indicating that these three compounds may be stimulating hydroquinone metabolism by a common mechanism. Interestingly, the stimulation of peroxidase-dependent hydroquinone metabolism by other phenolic compounds was pH-dependent, with the stimulating effect being higher under alkaline conditions. These results therefore suggest that the interaction of phenolic compounds, presumably by hydrogen-bonding, with the activity limiting distal amino acid residue(s) or with the ferryl oxygen of peroxidase may be an important contributing factor in the enhanced myeloperoxidase-dependent metabolism of hydroquinone in the presence of other phenolic compounds.     

Chemical and enzymatic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase. 2. Evidence for a substrate radical--enzyme complex and its reaction with nucleophiles.

Changes in the optical absorption spectrum of horseradish peroxidase, during the oxidation of o-dianisidine at pH 7.5, reveal an intermediate distinct from the previously described compounds I and II. The rate of decay of this new complex appeared to be rate limiting for the catalytic cycle, in this pH range, since imidazole, which augments the catalytic reaction, also enhanced the rate of decay of this complex. Nitrogenous compounds reportedly unable to ligate to hemes, such as 2-methylimidazole and benzimidazole, were nevertheless capable of augmenting the HRP-catalyzed rate of oxidation of o-dianisidine. The activity of nitrogenous compounds, in this regard, appeared to be a function of their nucleophilicity and was sensitive to steric factors but relatively free of a deuterium solvent isotope effect. The data presented in this and in the preceding paper [Claiborne, A., & Fridovich, I. (1979) Biochemistry 18 (preceding paper in this issue)] lead to the suggestion that the nucleophile-responsive intermediate is an enzyme--dianisidine radical complex and that abstraction of the second electron from the bound radical is facilitated by binding of nitrogenous nucleophiles.     

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.