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PubMed1976

Peroxidase-catalyzed halogenation.

Peroxidase-catalyzed halogenation reactions have been established as being important in the biosynthesis of the hormone thyroxine and in biological defense mechanisms. Recently these reactions have been recognized as valuable tools for the study of proteins as well as their arrangement in macromolecular structures. The pathways of peroxidase catalyses can be accommodated within the framework of the classical Chance-George mechanism. This implies that the initial steps of the reaction invariably involve oxidation of peroxidases by peroxides--and that the resulting derivative, compound I, is the oxidant of the halide ions. Such reactions may result either in the formation of hypohalous acids, or in halogenation of the enzyme apoprotein, followed by transhalogenation to substrate for halogenation. Chloro- and myeloperoxidases catalyze oxidation of all halide ions, except F-; oxidation of bromide and iodide is mediated by lactoperoxidase, but horseradish peroxidase only oxidizes iodide. All of the above enzymes except horseradish will oxidize the pseudo halide thiocyanate. The origins of this differentiation remain to be defined, but they presumably reflect significant variation in oxidation potential of different peroxidase-peroxide derivatives, rather than constraints on the peroxidase-donor interactions. As pointed out above, halogenation of the amino acids tyrosine and histidine or these residues in proteins can take place on the enzyme. This makes lactoperoxidase-catalyzed iodination selective. The amino acid residues in proteins that are iodinated depend not only on reactivity of the amino acid residue but also on its geometric location. Thus lactoperoxidase-catalyzed iodination can be a useful tool in the study of protein structure and function. It is also useful in establishing the geometric position of proteins within macromolecular structures. Thyroid peroxidase catalyzes iodination of thyroglobulin and is involved in a second important step, the coupling of the iodotyrosines to form thyroxine or triiodothyronine. A proposed mechanism for this reaction suggests that the oxidation is mediated by the iodoenzyme derivative mentioned above followed by a prototropic rearrangement and scission to form the ether bound of thyronine and a serine residue on thyroglobulin.

Amino Acids↗

PubMed1983

A peroxidase-coupled method for the colorimetric determination of serum triglycerides.

We describe an enzymatic method for rapid, precise measurement of serum triglycerides with use of sample:reagent ratios as large as 1:200. Hydrolysis of triglycerides is catalyzed by lipase to produce glycerol and free fatty acids. The glycerol generated is then phosphorylated by adenosine 5'-triphosphate in the presence of glycerol kinase. Oxidation of the resulting glycerol 3-phosphate to produce hydrogen peroxide is catalyzed by L-alpha-glycerophosphate oxidase. An intense red chromogen is produced by the peroxidase-catalyzed coupling of 4-aminoantipyrene and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate with hydrogen peroxide. This sensitive chromogen system not only permits use of unusually small sample volumes, it also facilitates a linear response to serum triglyceride concentrations up to at least 10 g/L while displaying good Ringbom (measure of accuracy) characteristics.

Adenosine Triphosphate↗

PubMed1995

Primary structure and characterization of the vanadium chloroperoxidase from the fungus Curvularia inaequalis.

Using reverse transcription of messenger RNA followed by amplification using the polymerase chain reaction, three overlapping cDNA fragments encompassing the encoding sequence of the vanadium chloroperoxidase from the fungus Curvularia inaequalis were isolated and sequenced. The sequence was confirmed by DNA sequence analysis of genomic DNA. The deduced amino acid sequence predicts a protein of 609 residues with a mass of 67488 Da. Competitive reverse-transcription polymerase chain reaction analysis indicates that vanadium chloroperoxidase expression takes place in the secondary-growth phase initiated by nutrient depletion. Southern-blot analysis of genomic DNA indicates that there is only a single gene encoding the vanadium chloroperoxidase and that no isoenzymes are present. The N-terminal amino acid residue was blocked and could not be determined by amino acid sequencing, although evidence is presented showing that the N-terminal region starts very close to the first encoded methionine residue. Although the vanadium chloroperoxidase is secreted, it was not possible to assign a leader peptide. The protein contains two putative N-glycosylation sites but experiments indicate that the protein is non-glycosylated. Two cysteine residues are present in the protein both as free thiols: no disulphide bridging was found. Metal analysis revealed that iron, copper, and calcium do not constitute part of the protein. Zinc was found at a ratio of 0.3 +/- 0.04 mol/mol protein. Boiling and subsequent SDS/PAGE of the protein sample showed a typical degradation pattern of the enzyme. Amino acid sequence analysis of the resulting peptides showed that the cleavage took place at Asp-Pro bonds of which six are located throughout the protein. No sequence similarity with other known peroxidases was found except for one small region, sharing limited similarity with bacterial haloperoxidases and other alpha/beta-hydrolase-fold enzymes. In the case of the bacterial bromoperoxidases from this group, a methionine located in this region was suggested to have a role in catalysis. Methionine, however, was not involved in the catalysis of the vanadium chloroperoxidase.

Amino Acid Sequence↗

PubMed1991

Sensitive immunometric assays for secretory peroxidase and myeloperoxidase in human saliva.

We have developed specific immunoassays for secretory peroxidase (SP) and for myeloperoxidase (MP) (polymorphonuclear leukocyte-derived peroxidase) in human saliva. Antibodies against SP and MP were produced using bovine milk lactoperoxidase (LP) and human MP as the immunogens, respectively. The methods developed are non-isotopic immunometric assays using biotinylated antibodies and avidin-enzyme conjugate. The detection limit was 0.1 ng/ml and the performance time less than 3 h for both assays. The determination ranges were 0.5-100 ng LP (SP)/ml and 0.5-200 ng MP/ml with intra- and interassay CVs of 4.3% and 15.6% for SP and 3.7% and 10.8% for MP, respectively. The mean analytical recoveries were 108.9% (SP) and 91.5% (MP). These assays correlated well (r = 0.849-0.871) with the colorimetric assays based on the oxidation of thiocyanate or chloride by peroxidases. However, compared to the colorimetric methods the new immunometric assays are much more sensitive and specific for salivary SP and MP. The assays are also more rapid since extensive dialysis to remove endogenous thiocyanate is not required.

Adult↗

PubMedDate not supplied

Catalase activity of chloroperoxidase and its interaction with peroxidase activity.

The catalatic activity of chloroperoxidase (CPO) was demonstrated to exhibit saturation kinetics under steady-state conditions, which were not observed with catalase under comparable conditions. Results were obtained using reaction mixtures of CPO and H2O2 at pH 6.2, rapid spectral scan and single wavelength measurements, and transient- and steady-state reaction conditions. The observed rectangular hyperbolae (measurement of rates of disappearance of H2O2 and appearance of O2) could be fit quantitatively to [equation: see text] where v is rate of O2 evolution, [CPO]0 is total enzyme concentration, B1 = (9 +/- 1) x 10(2) s-1, and B2 = (3.3 +/- 0.4) x 10(-3) M. The results indicated formation of a complex of compound I (CPO-I) and H2O2, which dissociated to native CPO, O2, and H2O with a rate constant of (9 +/- 1) x 10(2) s-1. The determination of the peroxidatic activity of CPO was performed using demethylation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) under steady-state conditions. Attempts to determine Michaelis-Menten constants for the substrates TMPD and H2O2 gave rise to apparently anomalous data. Our data showed that the modified ping-pong mechanism established for horseradish peroxidase is applicable to the peroxidatic reaction catalyzed by chloroperoxidase. Both peroxidatic and catalatic reactions occurred in the reaction system containing H2O2, a reducing substrate, and CPO. A combined reaction mechanism was proposed for CPO-catalyzed reactions in which the modified ping-pong mechanism was applicable for the peroxidatic reactions and the formation of a CPO-I-H2O2 complex occurred for the catalatic reaction.

Catalase↗

PubMed1988

Probing structure-function relations in heme-containing oxygenases and peroxidases.

Structural factors that influence functional properties are examined in the case of four heme enzymes: cytochrome P-450, chloroperoxidase, horseradish peroxidase, and secondary amine mono-oxygenase. The identity of the axial ligand, the nature of the heme environment, and the steric accessibility of the heme iron and heme edge combine to play major roles in determining the reactivity of each enzyme. The importance of synthetic porphyrin models in understanding the properties of the protein-free metal center is emphasized. The conclusions described herein have been derived from studies at the interface between biological and inorganic chemistry.

Catalysis↗

PubMed2004

Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes.

Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H(2)O(2) (2 mM) for optimum activity. The apparent K(m) values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H(2)O(2) were 37, 298, 1,001, 2,367 and 1,313 microM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.

Agaricales↗

PubMed2000

Oxidation reactions catalyzed by vanadium chloroperoxidase from Curvularia inaequalis.

Vanadium haloperoxidases have been reported to mediate the oxidation of halides to hypohalous acid and the sulfoxidation of organic sulfides to the corresponding sulfoxides in the presence of hydrogen peroxide. However, traditional heme peroxidase substrates were reported not to be oxidized by vanadium haloperoxidases. Surprisingly, we have now found that the recombinant vanadium chloroperoxidase from the fungus Curvularia inaequalis catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), a classical chromogenic heme peroxidase substrate. The enzyme mediates the oxidation of ABTS in the presence of hydrogen peroxide with a turnover frequency of 11 s(-1) at its pH optimum of 4.0. The Km of the recombinant enzyme for ABTS was observed to be approximately 35 microM at this pH value. In addition, the bleaching of an industrial sulfonated azo dye, Chicago Sky Blue 6B, catalyzed by the recombinant vanadium chloroperoxidase in the presence of hydrogen peroxide is reported.

Chloride Peroxidase↗

PubMed1989

Peroxyl, alkoxyl, and carbon-centered radical formation from organic hydroperoxides by chloroperoxidase.

The decomposition of organic hydroperoxides as catalyzed by chloroperoxidase was investigated with electron spin resonance (ESR) spectroscopy. Tertiary peroxyl radicals were directly detected by ESR from incubations of tert-butyl hydroperoxide or cumene hydroperoxide with chloroperoxidase at pH 6.4. Peroxyl, alkoxyl, and carbon-centered free radicals from tertiary hydroperoxide/chloroperoxidase systems were successfully trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide, whereas alkoxyl radicals were not detected in the ethyl hydroperoxide/chloroperoxidase system. The carbon-centered free radicals were further characterized by spin-trapping studies with tert-nitrosobutane. Oxygen evolution measured by a Clark oxygen electrode was detected for all the hydroperoxide/chloroperoxidase systems. The classical peroxidase mechanism is proposed to describe the formation of peroxyl radicals. In the case of tertiary peroxyl radicals, their subsequent self-reactions result in the formation of alkoxyl free radicals and molecular oxygen. beta-Scission and internal hydrogen atom transfer reactions of the alkoxyl free radicals lead to the formation of various carbon-centered free radicals. In the case of the primary ethyl peroxyl radicals, decay through the Russell pathway forms molecular oxygen.

Benzene Derivatives↗

PubMed1994

The chloroperoxidase-catalyzed oxidation of phenols. Mechanism, selectivity, and characterization of enzyme-substrate complexes.

The reactivity of a series of para-substituted phenolic compounds in the peroxidation catalyzed by chloroperoxidase was investigated, and the results were interpreted on the basis of the binding characteristics of the substrates to the active site of the enzyme. Marked selectivity effects are observed. These operate through charge, preventing phenolic compounds carrying amino groups on the substituent chain to act as substrates for the enzyme, and through size, excluding potential substrates containing bulky substituents to the phenol nucleus. Also, chiral recognition is exhibited by chloroperoxidase in the oxidation of N-acetyltyrosine, where only the L isomer is oxidized. Kinetic measurements show that, in general, the efficiency of chloroperoxidase in the oxidation of phenols is lower than that of horseradish peroxidase. Paramagnetic NMR spectra and relaxation rate measurements of chloroperoxidase-phenol complexes are consistent with binding of the substrates close to the heme, in the distal pocket, with the phenol group pointing toward the iron atom. On the other hand, phenolic compounds which are not substrates for chloroperoxidase bind to the enzyme with a much different disposition, with the phenol group very distant from the iron and probably actually outside the active-site cavity.

Catalysis↗

PubMed1979

Antithyroid and antiperoxidase activity of tropolone and 3-hydroxy-4-pyrone.

Tropolone (TR) and 3-hydroxy-4-pyrone were investigated for antithyroid activity following the finding that the 2-hydroxy-oxo pyridine, 3-hydroxy-4(1H)-pyridone (DHP, I), is goitrogenic. Both compounds inhibited the thyroidal uptake of radioiodine in rats and resembled the thioamide drugs in inhibiting the organic binding of iodine by the thyroid gland rather than the trapping of iodide, but were weaker binding inhibitors than 6-methyl-2-thiouracil (MeTU). Both compounds also inhibited the iodination of bovine serum albumin and thyroglobulin, catalyzed by thyroidperoxidase (TPO), lactoperoxidase (LPO), chloroperoxidase (CPO) and horseradish peroxidase (HPO) in vitro. The inhibitory effect of TR but not that of 3-hydroxy-4-pyrone was antagonized by ferrous ions. When fed to mice at levels of intake expected to produce goitre both compounds were toxic and caused severe liver damage. Thyroid enlargement was not observed in any of these feeiding experiments, but the thyroids of mice fed 0.1% TR showed moderate hyperplasia. It was concluded that both compounds are weakly goitrogenic. Hyperactivity was observed in the mice fed TR which may be associated with inhibition of catechol methyl transferase (COMT).

Animals↗

PubMed1985

Preparation and properties of ferrous chloroperoxidase complexes with dioxygen, nitric oxide, and an alkyl isocyanide. Spectroscopic dissimilarities between the oxygenated forms of chloroperoxidase and cytochrome P-450.

Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR-silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes.

Chloride Peroxidase↗

PubMed1989

EDTA inhibits peroxidase-catalyzed iodide oxidation through interaction at the iodide binding site.

EDTA inhibits the formation of I3- from iodide catalysed by various pure peroxidases. The inhibition is concentration-dependent and chloroperoxidase (CPO) is more sensitive than horseradish peroxidase (HRP) and lactoperoxidase (LPO). EDTA is more active than EGTA or other biological chelators tested. Zn2+, Mn2+ and Co2+ are equally active in reversing the effect of EDTA on both CPO and HRP almost completely, but ineffective in the case of LPO. The effect of EDTA on HRP can be reversed by a higher concentration of iodide but not by H2O2. EDTA causes a hypsochromic change in the absorption of the Soret band of HRP at 402 nm, and iodide can reverse this effect. EDTA can effectively displace radioiodide specifically bound to HRP. It is suggested that EDTA inhibits iodide oxidation by interacting at the iodide binding site of the HRP.

Chloride Peroxidase↗

PubMed2006

Resonance Raman spectroscopy of oxoiron(IV) porphyrin pi-cation radical and oxoiron(IV) hemes in peroxidase intermediates.

The catalytic cycle intermediates of heme peroxidases, known as compounds I and II, have been of long standing interest as models for intermediates of heme proteins, such as the terminal oxidases and cytochrome P450 enzymes, and for non-heme iron enzymes as well. Reports of resonance Raman signals for compound I intermediates of the oxo-iron(IV) porphyrin pi-cation radical type have been sometimes contradictory due to complications arising from photolability, causing compound I signals to appear similar to those of compound II or other forms. However, studies of synthetic systems indicated that protein based compound I intermediates of the oxoiron(IV) porphyrin pi-cation radical type should exhibit vibrational signatures that are different from the non-radical forms. The compound I intermediates of horseradish peroxidase (HRP), and chloroperoxidase (CPO) from Caldariomyces fumago do in fact exhibit unique and characteristic vibrational spectra. The nature of the putative oxoiron(IV) bond in peroxidase intermediates has been under discussion in the recent literature, with suggestions that the Fe(IV)O unit might be better described as Fe(IV)-OH. The generally low Fe(IV)O stretching frequencies observed for proteins have been difficult to mimic in synthetic ferryl porphyrins via electron donation from trans axial ligands alone. Resonance Raman studies of iron-oxygen vibrations within protein species that are sensitive to pH, deuteration, and solvent oxygen exchange, indicate that hydrogen bonding to the oxoiron(IV) group within the protein environment contributes to substantial lowering of Fe(IV)O frequencies relative to those of synthetic model compounds.

Chloride Peroxidase↗

PubMed1991

Peroxidase-catalyzed bromination of tyrosine, thyroglobulin, and bovine serum albumin: comparison of thyroid peroxidase and lactoperoxidase.

A recent paper (Buchberger, W., 1988, J. Chromatogr. 432, 57) on lactoperoxidase-catalyzed bromination of tyrosine and thyroglobulin stated, without evidence, that thyroid peroxidase (TPO) is able to use bromide as a substrate. This was in disagreement with unpublished experiments previously performed in this laboratory, and we undertook, therefore, to examine this subject further. Highly purified porcine TPO was compared with lactoperoxidase (LPO) and chloroperoxidase (CPO) for ability to catalyze bromination of tyrosine, thyroglobulin, and bovine serum albumin (BSA). The incubation mixture contained 50-100 nM peroxidase, 10-500 microM 82Br-, tyrosine (150 microM), thyroglobulin (0.3 or 1 microM), or BSA (7.5 microM), and a source of H2O2. The latter was either generated by glucose (1 mg/ml)-glucose oxidase (0.5 or 1 micrograms/ml), or added initially as a bolus (100 microM). With TPO, formation of organically bound 82Br was undetectable under all conditions in the pH range 5.4-7.0. Lactoperoxidase and CPO, on the other hand, displayed considerable brominating activity. Lactoperoxidase was much more active at pH 5.4 than at pH 7.0 and was more active with BSA as acceptor than with tyrosine or thyroglobulin. The distribution of 82Br among the various amino acids in LPO-brominated thyroglobulin and BSA was determined by HPLC. As expected, monobromotyrosine and dibromotyrosine together comprised the greatest part of the bound 82Br. However, a surprisingly high percentage (20-25%) was present as monobromohistidine. Evidence was also obtained for the presence of a small percentage of the bound 82Br as tetrabromothyronine. Peroxidase-catalyzed bromination probably depends on the oxidation of Br- to Br+ by the Compound I form of the enzyme. Since oxidation of Br- to Br+ requires a stronger oxidant than oxidation of I- to I+, our results suggest that Compound I of LPO and of CPO has a higher oxidation potential than Compound I of TPO. In vivo experiments with rats on a low iodine diet injected with 82Br- showed that even under conditions of high stimulation by thyrotropic hormone, there is negligible formation of organic bromine in the thyroid. Measurements of thyroid:serum concentration ratios for 82Br- in similar rats provided no evidence that Br- is a substrate for the iodide transport system of the thyroid.

Animals↗

PubMed1992

Mechanism of enantioselective oxygenation of sulfides catalyzed by chloroperoxidase and horseradish peroxidase. Spectral studies and characterization of enzyme-substrate complexes.

The binding of a series of alkyl aryl sulfides to chloroperoxidase (CPO) and horseradish peroxidase (HRP) has been investigated by optical difference spectroscopy, circular dichroism, paramagnetic NMR spectroscopy, and NMR relaxation measurements. The data are consistent with binding of the sulfides in the distal side of the heme pocket with CPO and near the heme edge with HRP. A linear correlation between the binding constants of para-substituted sulfides to CPO and the Taft sigma I parameter suggests that these substrates act as donors in donor-acceptor complexes involving some residue of the protein chain. Spectral studies during turnover show that high enantioselectivity in the CPO-catalyzed oxidation of sulfides results from a reaction pathway that does not involve the accumulation of compound II enzyme intermediate.

Catalysis↗

PubMed1985

Peroxidase-catalyzed N-demethylation reactions: deuterium solvent isotope effects.

The effect of D2O on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase was investigated in order to assess the roles of exchangeable hydrogens in the demethylation reaction. The initial rate of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide exhibited a pL optimum (where L denotes H or D) of 4.5 in both H2O and D2O. The solvent isotope effect on the initial rate of the chloroperoxidase-catalyzed demethylation reaction was independent of pL, suggesting that the solvent isotope effect is not due to a change in the pK of a rate-controlling ionization in D2O. The solvent isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction was 3.66 +/- 0.62. In contrast, the solvent isotope effect on the Vmax for the horseradish peroxidase catalyzed demethylation reaction was approximately 1.5 with either ethyl hydroperoxide or hydrogen peroxide as the oxidant, indicating that the exchange of hydrogens in the enzyme and hydroperoxide for deuterium in D2O has little effect on the rate of the demethylation reaction. The solvent isotope effect on the Vmax/KM for ethyl hydroperoxide in the chloroperoxidase-catalyzed demethylation reaction was 8.82 +/- 1.57, indicating that the rate of chloroperoxidase compound I formation is substantially decreased in D2O. This isotope effect is suggested to arise from deuterium exchange of the hydroperoxide hydrogen and of active-site residues involved in compound I formation. A solvent isotope effect of 2.96 +/- 0.57 was observed on the Vmax/KM for N,N-dimethylaniline in the chloroperoxidase-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride Peroxidase↗

PubMed1991

Heme-protein fission under nondenaturing conditions.

Slow heme transfer from horseradish peroxidases C2 and A2, cytochrome c peroxidase, chloroperoxidase, and leghemoglobins to a heme acceptor protein, apomyoglobin, has been studied under mild conditions. The reaction is best described as heme release into water followed by quick engulfment by apomyoglobin. The energetics of the activated process are large and interpreted as connected to both polypeptide motions during release and the ordering of water around the heme during solvation. The free energy required to break the iron(III)-ligand 5 (L5) bond is a minor but crucial portion of the activation free energy. Donor-acceptor protein interactions are not involved in the transfer. Fast heme release from inactive protein has also been observed. Apoprotein recombination with porphyrins and hemes suggest that this lack of activity is a result of Fe-L5 bond breaking.

Apoproteins↗