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PubMed1992

1H NMR investigation of manganese peroxidase from Phanerochaete chrysosporium. A comparison with other peroxidases.

1H NMR spectra at 200- and 600-MHz of manganese peroxidase from Phanerochaete chrysosporium and of its cyanide derivative are reported. The spectrum of the native protein is very similar to that of other peroxidases. The assignment of the spectrum of the cyanide derivative has been performed through 1D NOE, 2D NOESY, and COSY experiments. This protein is very similar to lignin peroxidase, the only meaningful difference being the shift of H delta 2 of the proximal histidine. The spectra of the cyanide derivative of these two proteins are compared with those of horseradish peroxidase and cytochrome c peroxidase. The shift pattern of the protons of the proximal histidine is discussed relative to the structural properties which affect the Fe3+/Fe2+ redox potential.

Chemical Phenomena↗
PubMed1990

Combination of the peroxidase anti-peroxidase (PAP)- and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.

A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step--Incubation of the tissue sections with the monoclonal primary antibodies; Second step--biotinylated anti-rat or anti-mouse IgG; Third step--monoclonal PAP complex; Fourth step--ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step--the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4- and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Peroxidase-catalyzed oxidation of ascorbate. Structural, spectroscopic and mechanistic correlations in ascorbate peroxidase.

Ascorbate-dependent peroxidase activity was first reported in 1979 (Groden and Beck, 1979; Kelly and Latzko, 1979) and ascorbate peroxidase (APX) is, therefore, a relative newcomer to the peroxidase field--horseradish (HRP) and cytochrome c (CcP) peroxidases were, for example, first identified in 1903 (Bach and Chodat, 1903) and 1940 (Altschul et al., 1940) respectively. The APX area was reviewed by Dalton in 1991 (Dalton, 1991): at that time, there was very little detailed kinetic, spectroscopic or functional information available and no structural information had been published. Since 1991, there have been some major advances in the field, most notably with the publication, in 1995, of the first crystal structure for an APX enzyme (Patterson and Poulos, 1995). This information, together with the availability of new recombinant expression systems (Yoshimura et al., 1998; Caldwell et al., 1998; Dalton et al., 1996; Patterson and Poulos, 1994), served as a catalyst for the publication of new functional and spectroscopic data and has meant these data could be sensibly rationalized at the molecular level. The aim of this review is to summarize the more recent advances in the APX area and, as far as possible, to draw comparisons with other, more well-characterized peroxidases. The review will concentrate on the ways in which structural, spectroscopic and mechanistic information have been used in a complementary way to provide a more detailed picture of APX catalysis. The more biological and physiological aspects of APX enzymes have been previously covered in a comprehensive manner (Dalton, 1991) and will not, therefore, be dealt with in detail here.

Ascorbate Peroxidases↗
PubMed1997

Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase.

BACKGROUND: Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase. The Mn-binding site in MnP is of particular interest. Oxidation of Mn(II) to Mn(III) is a key step in the biodegradation of lignin, a complex phenylpropanoid polymer, as well as many aromatic pollutants. Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese. Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure and function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds. RESULTS: Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (Gly41-->Glu, Val45-->Glu, His181-->Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity. Titrating MnSO4 into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP. The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CCP were also similar to MnP. Titration with MnSO4 resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP. None of the spectral features were observed in wild-type CcP. In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP. CONCLUSIONS: A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP. The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.

Binding Sites↗
PubMed1982

Oxidation of indole-3-acetic acid by peroxidase: involvement of reduced peroxidase and compound III with superoxide as a product.

Kinetic and spectral data establish that peroxidase may oxidize indole-3-acetic acid by either of two pathways depending on the enzyme/substrate ratio. When relatively low enzyme/substrate ratios are employed, the oxidation proceeds through a reduced peroxidase in equilibrium compound III shuttle. Conversely, peroxidase operates through the conventionally accepted pathway involving native enzyme and compounds I and II only when high enzyme/substrate ratios are used. Compound III, a specific oxidase, constitutes the dominant steady-state form of peroxidase when the reduced peroxidase in equilibrium compound III shuttle is operational. Activation of this shuttle also produces a flux of superoxide anion radical at the expense of molecular oxygen. Thus, important biological consequences may follow activation of this shuttle under physiological conditions.

Catalase↗
PubMed2003

Mechanism of horseradish peroxidase inactivation by benzhydrazide: a critical evaluation of arylhydrazides as peroxidase inhibitors.

Many compounds are oxidized by haem enzymes, such as peroxidases and cytochromes P450, to highly reactive intermediates that function as enzyme inactivators. To evaluate the potential of arylhydrazides as selective metabolically activated peroxidase inhibitors, the mechanism of HRPC (horseradish peroxidase isoenzyme C) inhibition by BZH (benzhydrazide) was investigated in detail. No oxygen consumption was detected in BZH solutions at pH 7.0-12.0, but addition of HRPC resulted in significant O2 uptake above pH 8.0, indicating that the enzyme catalyses BZH oxidation. Addition of H2O2 to HRPC plus BZH activates the latter as an inhibitor. This involves the three-electron oxidation of BZH in one-electron steps by the peroxidase catalytic intermediates, Compounds I and II, to produce a benzoyl radical that covalently alters the active site and inhibits peroxidase activity. Alternatively, the benzoyl radical could be produced by di-imide (NH=NH) elimination from the BZH radical. Production of Compound III (oxyperoxidase) followed by p-670 (m/z =583, biliverdin-like derivative) was observed for HRPC incubated with excess H2O2, and the addition of BZH resulted in an increase in the rate of p-670 production. BZH is an inefficient inhibitor of HRPC with a K(I) of 80 muM, an apparent inactivation rate constant (k(inact)) of 0.035 min(-1), and an IC50 of 1.0 mM. This prompted the investigation of HRPC inactivation by a series of related arylhydrazides with known binding affinities for HRPC. The hydrazide with the highest affinity (2-naphthoichydrazide; K(d)=5.2 muM) was also found to be the most effective inhibitor with K(I), k(inact) and IC50 values of 14 muM, 0.14 min(-1) and 35 muM, respectively.

Catalysis↗
PubMed1990

Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes.

Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The present study investigated the possibility that other peroxidative and oxidative enzymes known to catalyse one-electron oxidations may also oxidize non-phenolics to cation-radical intermediates and that this ability is related to the redox potential of the substrate. Lignin peroxidase from the fungus Phanerochaete chrysosporium, horseradish peroxidase (HRP) and laccase from the fungus Trametes versicolor were chosen for investigation with methoxybenzenes as a homologous series of substrates. The twelve methoxybenzene congeners have known half-wave potentials that differ by as much as approximately 1 V. Lignin peroxidase oxidized the ten with the lowest half-wave potentials, whereas HRP oxidized the four lowest and laccase oxidized only 1,2,4,5-tetramethoxybenzene, the lowest. E.s.r. spectroscopy showed that this congener is oxidized to its cation radical by all three enzymes. Oxidation in each case gave the same products: 2,5-dimethoxy-p-benzoquinone and 4,5-dimethoxy-o-benzoquinone, in a 4:1 ratio, plus 2 mol of methanol for each 1 mol of substrate. Using HRP-catalysed oxidation, we showed that the quinone oxygen atoms are derived from water. We conclude that the three enzymes affect their substrates similarly, and that whether an aromatic compound is a substrate depends in large part on its redox potential. Furthermore, oxidized lignin peroxidase is clearly a stronger oxidant than oxidized HRP or laccase. Determination of the enzyme kinetic parameters for the methoxybenzene oxidations demonstrated further differences among the enzymes.

Benzene Derivatives↗
PubMed2000

The putative glutathione peroxidase gene of Plasmodium falciparum codes for a thioredoxin peroxidase.

A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was active with H(2)O(2) and organic hydroperoxides but, unlike phospholipid hydroperoxide glutathione peroxidase, not with phosphatidylcholine hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism with infinite V(max) and K(m) when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum (Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K(m) of 10 microm and a k(1)' of 0.55 s(-)1. The apparent k(1)' for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 x 10(4) m(-1) s(-1), 3.3 x 10(3) m(-1) s(-1), and 2.5 x 10(3) m (-1) s(-1), respectively. k(2)' for reduction by autologous thioredoxin is 5.4 x 10(4) m(-1) s(-1) (21.2 m(-1) s(-1) for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.

Amino Acid Sequence↗
PubMed2003

Rapid formation of compound II and a tyrosyl radical in the Y229F mutant of Mycobacterium tuberculosis catalase-peroxidase disrupts catalase but not peroxidase function.

Catalase-peroxidases (KatG), which belong to Class I heme peroxidase enzymes, have high catalase activity and substantial peroxidase activity. The Y229F mutant of Mycobacterium tuberculosis KatG was prepared and characterized to investigate the functional role of this conserved residue unique to KatG enzymes. Purified, overexpressed KatG[Y229F] exhibited severely reduced steady-state catalase activity while the peroxidase activity was enhanced. Optical stopped-flow experiments showed rapid formation of Compound (Cmpd) II (oxyferryl heme intermediate) in the reaction of resting KatG[Y229F] with peroxyacetic acid or chloroperoxybenzoic acid, without detectable accumulation of Cmpd I (oxyferryl heme pi-cation radical intermediate), the latter being readily observed in the wild-type enzyme under similar conditions. Facile formation of Cmpd III (oxyferrous enzyme) also occurred in the mutant in the presence of micromolar hydrogen peroxide. Thus, the lost catalase function may be explained in part because of formation of intermediates that do not participate in catalatic turnover. The source of the reducing equivalent required for generation of Cmpd II from Cmpd I was shown by rapid freeze-quench electron paramagnetic resonance spectroscopy to be a tyrosine residue, just as in wild-type KatG. The kinetic coupling of radical generation and Cmpd II formation was shown in KatG[Y229F]. Residue Y229, which is a component of a newly defined three amino acid adduct in catalase-peroxidases, is critically important for protecting the catalase activity of KatG.

Bacterial Proteins↗
PubMed2005

Substrate specificity, localization, and essential role of the glutathione peroxidase-type tryparedoxin peroxidases in Trypanosoma brucei.

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical cysteine homologues of the classical selenocysteine-containing glutathione peroxidases. Although one of the sequences, peroxidase III, carries both putative mitochondrial and glycosomal targeting signals, the proteins are detectable only in the cytosol and mitochondrion of mammalian bloodstream and insect procyclic T. brucei. The enzyme is a trypanothione/tryparedoxin peroxidase as are the 2 Cys-peroxiredoxins of the parasite. Hydrogen peroxide, thymine hydroperoxide, and linoleic acid hydroperoxide are reduced with second order rate constants of 8.7 x 10(4), 7.6 x 10(4), and 4 x 10(4) m(-1) s(-1), respectively, and represent probable physiological substrates. Phosphatidylcholine hydroperoxide is a very weak substrate and, in the absence of Triton X-100, even an inhibitor of the enzyme. The substrate preference clearly contrasts with that of the closely related T. cruzi enzyme, which reduces phosphatidylcholine hydroperoxides but not H(2)O(2). RNA interference causes severe growth defects in bloodstream and procyclic cells in accordance with the peroxidases being essential in both developmental stages. Thus, the cellular functions of the glutathione peroxidase-type enzymes cannot be taken over by the 2 Cys-peroxiredoxins that also occur in the cytosol and mitochondrion of the parasite.

Animals↗
PubMed1978

[Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase].

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.

Acylation↗
PubMed1996

Strategies for the suppression of peroxidase gene expression in tobacco. II. In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs.

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expression in planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the lignin-forming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40-80% was seen in the T0 and T1 populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzs in vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.

Agrobacterium tumefaciens↗
PubMed1985

Enzymatic reduction of 5-phenyl-4-pentenyl-hydroperoxide: detection of peroxidases and identification of peroxidase reducing substrates.

5-Phenyl-4-pentenyl-hydroperoxide (PPHP) is reduced to 5-phenyl-4-pentenyl-alcohol (PPA) by plant and animal peroxidases in the presence of reducing substrates. PPHP and PPA are rapidly isolated with solid phase extraction, separated by isocratic reverse-phase high-performance liquid chromatography, and quantitated with a fixed-wave-length ultraviolet detector. The procedure described is suitable for detecting peroxide-reducing enzymes, determining the kinetic properties of heme- and non-heme-containing peroxidases, and evaluating oxidizable compounds as reducing substrates for peroxidases. Horseradish peroxidase (HRP) and phenol reduce PPHP with a Km for phenol of 252 microM and a turnover number of 1.05 X 10(4) min-1. Under similar conditions, the Km of HRP for PPHP is 18 microM in the oxidation of guaiacol. A series of 21 compounds was evaluated for the ability to serve as reducing substrates for HRP. The results indicate that the procedure described can not only identify compounds that are reducing substrates but also rank them for relative activity. This may provide a new method with which to identify novel antithrombotic, antimetastatic, or anti-inflammatory drugs as well as to detect and characterize mammalian peroxidases.

Alkenes↗
PubMed1994

Intimate relation between cyclooxygenase and peroxidase activities of prostaglandin H synthase. Peroxidase reaction of ferulic acid and its influence on the reaction of arachidonic acid.

The oxidation of ferulic acid by hydrogen peroxide catalyzed by prostaglandin H synthase follows a modified ping-pong irreversible mechanism, as is the case for classical peroxidases. The rate constant for the reaction of prostaglandin H synthase with hydrogen peroxide, determined from steady-state results, is (1.31 +/- 0.1) x 10(6) M-1 s-1, and for the reaction of prostaglandin H synthase-compound II with ferulic acid it is (5.5 +/- 0.3) x 10(6) M-1 s-1. Cyclooxygenase and peroxidase functions of prostaglandin H synthase were studied by comparing the initial rates of reaction of the cyclooxygenase substrate, arachidonic acid, and a peroxidase reducing substrate, ferulic acid, in mixtures of the two substrates. For both an equimolar ratio of arachidonic and ferulic acids and ferulic acid in excess of arachidonic acid a stimulation of the cyclooxygenase reaction is observed. The concentration of ferulic acid necessary to produce 50% stimulation of 0.2 mM arachidonic acid oxidation is 0.14 +/- 0.02 mM. A striking feature of our results is that prostaglandin H synthase catalyzes oxidation of the two substrates in a constant and fixed molar ratio of ferulic acid to arachidonic acid of 2:1, despite widely different starting concentrations. If arachidonic acid is in excess of ferulic acid, enzyme inactivation occurs. The results can be explained by an interconnected cyclooxygenase-peroxidase unbranched free radical mechanism in which arachidonic acid reacts with either the ferryl oxygen or the porphyrin pi-cation radical part of a conventional peroxidase compound I (a FeIV = O porphyrin pi-cation radical) and ferulic acid reacts with compound II (FeIV = O).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Hydrogen bond network in the distal site of peroxidases: spectroscopic properties of Asn70 --> Asp horseradish peroxidase mutant.

The distal His in peroxidases forms a hydrogen bond with the adjacent Asn, which is highly conserved among many plant and fungal peroxidases. Our previous work [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., & Morishima, I. (1996) Biochemistry 35, 14251-14258] has revealed that the replacement of Asn70 in horseradish peroxidase C (HRP) by Val (N70V) and Asp (N70D) discourages the oxidation activity for guaiacol, and the elementary reaction rate constants for the mutants was decreased by 10-15-fold. In order to delineate the structure-function relationship of the His-Asn couple in peroxidase activity, heme environmental structures of the HRP mutant, N70D, were investigated by CD, 1H NMR, and IR spectroscopies as well as Fe2+/Fe3+ redox potential measurements. While N70D mutant exhibited quite similar CD spectra and redox potential to those of native enzyme, the paramagnetic NMR spectrum clearly showed that the hydrogen bond between the distal His and Asp70 is not formed in the mutant. The disappearance of the splitting in the 1H NMR signal of heme peripheral 8-methyl group observed in 50% H2O/50% D2O solution of N70D-CN suggests that the hydrogen bond between the distal His and heme-bound cyanide is also disrupted by the mutation, which was supported by the low C-N vibration frequency and large dissociation constant of the heme-bound cyanide in the mutant. Together with the results from various spectroscopies and redox potentials, we can conclude that the improper positioning of the distal His induced the cleavages of the hydrogen bonds around the distal His, resulting in the substantial decrease of the catalytic activity without large structural alterations of the enzyme. The His-Asn hydrogen bond in the distal site of peroxidases, therefore, is essential for the catalytic activity by controlling the precise location of the distal His.

Asparagine↗
PubMed2000

The heme-independent manganese-peroxidase activity depends on the presence of the C-terminal domain within the Streptomyces reticuli catalase-peroxidase CpeB.

Streptomyces reticuli produces a heme-containing homodimeric enzyme (160 kDa), the catalase-peroxidase CpeB, which is processed to the enzyme CpeC during prolonged growth. CpeC contains four subunits of 60 kDa each that do not include the C-terminal portion of the progenitor subunits. A genetically engineered cpeB gene encodes a truncated subunit lacking 195 of the C-terminal amino acids; four of these subunits assemble to form the enzyme CpeD. Heme binds most strongly in CpeB, least in CpeD. The catalase-peroxidase CpeB and its apo-form (obtained after extraction of heme) catalyze the peroxidation of Mn(II) to Mn(III), independent of the presence or absence of the heme inhibitor KCN. CpeC and CpeD, in contrast, do not exhibit manganese-peroxidase activity. The data show for the first time that a bacterial catalase-peroxidase has a heme-independent manganese-peroxidase activity, which depends on the presence of the C-terminal domain.

Amino Acid Sequence↗
PubMed1992

Peroxidase and coupling activities of thyroid peroxidase in benign and malignant thyroid tumor tissues.

The coupling activity of thyroid peroxidase (TPO) in thyroid glands from patients with benign adenoma, papillary carcinoma, and diffuse goiter (Graves' disease) was measured for the first time, in addition to the peroxidase activity of these tissues. The peroxidase activity of TPO in the mitochondria-microsomes fraction was measured with guaiacol or iodide as the second substrate. In the case of papillary carcinoma, the mean protein-based specific activity obtained by the guaiacol assay was about 1/7 of that of diffuse goiter. The iodide oxidation activity of carcinoma was very low, about 1/25 [corrected] of that in diffuse goiter and 1/70 of that in adenoma. The peroxidase activity in adenoma was almost similar in the guaiacol oxidation assay and approximately one half in the iodide oxidation assay as compared with that in diffuse goiter. There was a close correlation between the guaiacol and iodide oxidation assays in individual patients with adenoma and diffuse goiter, but not in patients with papillary carcinoma. The coupling activity of TPO was measured with thyroglobulin purified from pooled toxic diffuse goiters and chemically iodinated to contain little additional T3 and T4. The specific coupling activity of TPO in mitochondria-microsomes from carcinoma was significantly lower (about 1/5) than that of diffuse goiter, and the activity in adenoma was not significantly different (about 1/2) from that of diffuse goiter. The data of coupling activities has a close correlation with that of peroxidase activities in individual patients with adenoma but not in patients with carcinoma. Based on these findings, the qualitative abnormality of TPO and its relation to the cold 123I scintigram in thyroid tumors are discussed.

Adenoma↗
PubMed2001

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions.

Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.

Binding Sites↗