E. A. Mareeva

Aerobic oxidation of indole-3-acetic acid catalysed by anionic and cationic peanut peroxidase

The catalytic properties of anionic and cationic peanut peroxidases with regards to the oxidation of indole-3-acetic acid (IAA) by molecular oxygen at low pH have been studied. Transient kinetic studies demonstrate that only cationic peroxidases (peanut and horseradish) but not anionic peroxidases (such as anionic tobacco and anionic peanut peroxidases) form a stable compound III in the course of IAA oxidation. The failure to observe inhibition in the presence of superoxide dismutase is consistent with the formation of compound III from a ternary complex comprising ferric enzyme, IAA and dioxygen at the initiation step. Product analysis by HPLC showed an enhanced rate of IAA oxidation in the presence of superoxide dismutase. Co-addition of superoxide dismutase and catalase demonstrates that this stimulation is not due to the formation of hydrogen peroxide. The correlation between initial rates of IAA degradation and product accumulation indicates that skatole hydroperoxide is a primary reaction product and indole-3-methanol is the product of its subsequent enzymatic reduction. The relative catalytic activities for IAA oxidation by tobacco:horseradish isoenzyme c:anionic peanut:cationic peanut peroxidase are 28:20:2:1.

Direct and Mediated Electron Transfer Catalyzed by Anionic Tobacco Peroxidase: Effect of Calcium Ions

The properties of anionic tobacco peroxidase (TOP) adsorbed on graphite electrode have been studied in direct and mediated electron transfer in a wall-jet flow injection system. The percentage of tobacco peroxidase molecules active in directelectron transfer is about 83%, which is higher than that for horeradish peroxidase (40–50%). This observation is explained in terms of the lower degree of glycosylation of TOP compared with horseradish peroxidase and, therefore, a reduced in terference from the oligosaccharide chains with direct electron transfer. Calcium ions cause an 11% drop in the reaction rate constant toward hydrogen peroxide. The detection limit of calcium chloride has been estimated as 5 m M. The results obtained by means of bioeletrochemistry, stopped-flow kinetics, and structural modeling provide evidence for the interaction between calcium cations and negatively charged residues at the distal domain (Glu-141, heme propionates, Asp-79, Asp-80) blocking the activesite. The observation that both soluble and immobilized enzyme under go conformational changes resulting in the blockade of the active site indicates that the immobilized enzyme preserves conformational flexibility. An even stronger suppressing effect of calcium ions on the rate constant for mediated electron transfer was observed. In the case of direct electron transfer, this couldmean that there is nodirect contact between the electrode and the active site of TOP. The electrons are shuttled from the active site to the surface of the electrode through electron transfer pathways in the protein globule that are sensitive to protein conformational changes.

Wild-type and mutant forms of recombinant horseradish peroxidase C expressed in Escherichia coli - Substrate specificity and stability under irradiation

Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41→ His and Phel43→ Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phel43→ Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals.The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2′-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and o-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for o-phenylene diamine.

Comparative study of the inactivation of horseradish peroxidase under the effect of H2O2 and ionizing radiation

The inactivation of native and recombinant horseradish peroxidase in the presence of hydrogen peroxide and under ionizing radiation was studied. The types of peroxidase activity differ in sensitivity towards the inactivating effect of H2O2: the activity in relation to the iodide ion is more stable than the activity in relation to ammonium 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonate) (ABTS) ando-phenylenediamine. Similar inactivation was observed in the course of the radiolysis of peroxidase. It was assumed that the initial period of peroxidase inactivation in the presence of hydrogen peroxide has a radical nature and is related to the generation of Superoxide radicals, which modify the protein moiety, resulting in the destruction of heme. The R-670 compound was not formed under the conditions studied. However, the E → EI transition occurred, depending on the radiation dose and the enzyme concentration.

Changes in substrate specificity of native and recombinant horseradish peroxidase under ionizing radiation

The homogeneous recombinant horseradish peroxidase preparation fromE. coli inclusion bodies exhibits higher specific activity towards ammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) than the native one. The differences in substrate specificity can be assigned to the native enzyme inactivation in the course of metabolic reactions in living plant cells, while the recombinant enzyme reconstructedin vitro completely realizes the original catalytic abilities. Application of the method of radiation-induced inactivation demonstrates the existence of different binding sites for the iodide anion. ABTS, phenol, and guaiacol and allows one to assume a common character of the binding sites of phenol ando-phenylenediamine.

Comparative studies of stability of native and recombinant horseradish peroxidase inactivated by radiation and other factors

Comparative studies of the inactivation of native and recombinant horseradish peroxidase in the course of an enzymatic reaction, at elevated temperatures and in a wide range of radiation doses, have been performed. The protective effect of the carbohydrate component of the native peroxidase providing for stabilization of the enzyme against various inactivating factors was demonstrated. It was proposed that radioactive inactivation is related to dysfunction in heme interaction with the protein component and to an increase in the conformational mobility around the active site of the enzyme.

Catalytic properties of Phe41→His mutant of horseradish peroxidase expressed inE. coli

The recombinant horseradish peroxidase and its single-point F41H mutant have been reactivated fromE. coli inclusion bodies. The influence of the mutation on the heme entrapment, stability and activity of the enzyme was demonstrated. The catalytic rate constants for H2O2 cleavage and ammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) oxidation decrease by two and one orders of magnitude, respectively. Unlike the wild-type recombinant horseradish peroxidase, the elimination of the ABTS oxidation product is not a rate-determining step for the mutant. The F41H replacement results in significant changes of kinetics of iodide ion oxidation. The reaction rate is linear to the concentrations of iodide, H2O2, and the enzyme. The results suggest the direct interaction of iodide with the porphyrin ring of the heme. The decrease in ABTS oxidation activity accompanied by retention of activity in iodide oxidation in the course of low-dosage radiolysis of the F41H mutant is additional evidence of the direct electron transfer from iodide to the heme, in contrast to ABTS oxidation, in which the electron transfer chain in the protein molecule is involved.

The role of tryptophan residues in radiation sensitivity of enzymes as exemplified in horseradish peroxidase

The stability of recombinant wild-type horseradish peroxidase and its tryptophan-less mutant Trp117Phe toward γ-radiation was studied. The absence of tryptophan in the enzyme molecule results in a certain stabilization, which is manifested as the absence of the initial drop in activity and appearance of a lag period for doses of up to 45 Gy. Contrary to the wild-type enzyme, the dose response of the mutant is almost independent of the nature of the substrate used to measure the catalytic activity; this indirectly indicates that Trp117 participates in the oxidation of substrates. Pretreatment of the wild-type recombinant enzyme with hydrogen peroxide destabilizes the enzyme towards irradiation, while the same procedure for the mutant enzyme has virtually no effect on the dose response curve. This suggests the modification of Trp117 in the oxidation of the native enzyme with H2O2 in the absence of electron-donor substrates, which is the modification of Trp171 in the recombinant lignin peroxidase.