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PubMed1994

Effects of chloride on the kinetics and stereochemistry of chloroperoxidase catalyzed oxidation of sulfides.

At pH 3, chloride dramatically influenced both the Km of chloroperoxidase (CPO) for methyl p-tolyl sulfide, which decreased, and its activity, which increased. The Km value changed from 75 microM in the absence of chloride to < or = 1.2 microM in > or = 0.9 mM chloride, and the kcat from 53 s-1 in 0 to 1750 s-1 in 50 mM halide. The kcat/Km value at 0.9 mM chloride was 414 microM -1s-1 compared to 0.7 microM-1s-1 in the absence of the halide. At pH 5, the activating effect was less pronounced. Chloride also acted as inhibitor versus hydrogen peroxide. The data are consistent with a reaction mechanism in which, on hand, chloride competes with hydrogen peroxide for the native enzyme and, on the other hand, activates sulfide oxidation by binding to CPO Compound I to give a CPO-chlorinating intermediate (EOCl-). However, contrary to what happened in the absence of chloride, where the oxidation was enantioselective and an oxygen atom of H2O2 was incorporated in the sulfoxide (from experiments with 18O-labeled H2O2), in the presence of the halide the oxidation was not enantioselective and there was no incorporation of oxygen from H2O2. The data suggest that sulfide oxidation takes place through an enzyme-generated freely dissociable oxidized halogen intermediate formed by the interaction of EOCl- with Cl-.

Chloride Peroxidase↗
PubMed2001

Synthetic active site analogues of heme-thiolate proteins. Characterization and identification of intermediates of the catalytic cycles of cytochrome P450cam and chloroperoxidase.

In view of recent results from different sources, the reaction mechanisms of two heme-thiolate proteins, cytochrome P450cam and chloroperoxidase (CPO), are discussed. In this context a mechanism of CPO is proposed which includes H2O2 cleavage, subsequent formation of compound I and the identification of two elusive intermediates. The HOCl adduct of the iron(III)porpyhrin is the catalytically competent Cl+ donor chlorinating activated C-H bonds of substrates bound to the enzyme. Pulse-EPR characterization of an enzyme model of the resting state of P450cam suggests a role of the electric field of the protein for stabilizing the low-spin state of the cofactor of the enzyme. It is further suggested that the same effect of the protein may trigger the reactivity of compound I such that both concerted and two-step reactions are feasible within the concept of a Two-State-Reactivity. This review emphasizes the value of synthetic enzyme models complementing investigations of the native proteins.

Binding Sites↗
PubMed1997

A nuclear paramagnetic relaxation study of the interaction of the cyclopentanedione substrate with chloroperoxidase.

Association of the physiological substrate cyclopentanedione (CPDO) with chloroperoxidase (CPO) was confirmed from both line-width and longitudinal relaxation time measurements of the proton NMR signals of CPDO. A dissociation constant of 33 mM for the enzyme-substrate complex was calculated from proton NMR relaxation experiments. Chloride ion does not affect the stability of the complex, but iodide ion greatly decreased the stability of the CPO-CPDO complex. Binding of cyanide ion to the heme iron center of CPO also decreased the stability of the CPO-CPDO complex. This result indicates the critical influence of perturbation in the heme active site on the affinity of CPO toward its substrate. Titration of CPO with CPDO revealed that CPDO does not bind directly to the heme iron of CPO. A distance of 7.1 A between the heme center of CPO and the protons of bound CPDO is deduced from the Solomon-Bloembergen relationship. This distance is 1 to 3 A shorter than those reported for the complexes formed between CPO and organic sulfide and unreactive phenol substrates, but the distance approximates that for reactive phenol substrates.

Binding Sites↗
PubMed1998

Isolation, characterization, and primary structure of the vanadium chloroperoxidase from the fungus Embellisia didymospora.

Here we describe the isolation, purification, and basic kinetic parameters of a vanadium type chloroperoxidase from the hyphomycete fungus Embellisia didymospora. The enzyme proved to possess similar high substrate affinities, a Km of 5 microM for a bromide, 1.2 mM for a chloride, and 60 microM for a hydrogen peroxide, as those of the vanadium chloroperoxidase from Curvularia inaequalis, although with lower turnover rates for both Cl- and Br-. Substrate bromide was also found to inhibit the enzyme, a feature subsequently also noted for the chloroperoxidase from C. inaequalis. The gene encoding this enzyme was identified using DNA Southern blotting techniques and subsequently isolated and sequenced. A comparison is made between this vanadium chloroperoxidase and that of the fungus C. inaequalis both kinetically and at the sequence level. At the primary structural level the two chloroperoxidases share 68% identity, with conservation of all active site residues.

Amino Acid Sequence↗
PubMed1998

The biosynthesis of differentiation-inducing factor, a chlorinated signal molecule regulating Dictyostelium development.

Differentiation-inducing factor (DIF)-1 is a chlorinated alkyl phenone released by developing Dictyostelium amoebae, which induces them to differentiate into stalk cells. A biosynthetic pathway for DIF-1 is proposed from labeling, inhibitor, and enzymological experiments. Cells incorporate 36Cl- into DIF-1 during development, showing that the chlorine atoms originate from chloride ions; peak incorporation is at the first finger stage. DIF-1 synthesis can be blocked by cerulenin, a polyketide synthase inhibitor, suggesting that it is made from a polyketide. This is most likely the C12 polyketide (2,4,6-trihydroxyphenyl)-1-hexan-1-one (THPH). Feeding experiments confirm that living cells can convert THPH to DIF-1. Conversion requires both chlorination and methylation of THPH, and enzymatic activities able to do this exist in cell lysates. The chlorinating activity, assayed using 36Cl-, is stimulated by H2O2 and requires both soluble and particulate components. It is specific for THPH and does not use this compound after O-methylation. The methyltransferase is soluble, uses S-adenosyl-L-methionine as a co-substrate, has a Km for dichloro-THPH of about 1.1 microM, and strongly prefers this substrate to close analogues. Both chlorinating and methyltransferase activities increase in development in parallel with DIF-1 production, and both are greatly reduced in a mutant strain that makes little DIF-1. It is proposed that DIF-1 is made by the initial assembly of a C12 polyketide skeleton, which is then chlorinated and methylated.

Animals↗
PubMed2006

Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate, and nitrate.

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 A. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.

Acetic Acid↗
PubMed1994

The stability and steady-state kinetics of vanadium chloroperoxidase from the fungus Curvularia inaequalis.

In this article we report on the steady-state kinetics of the chlorination and the stability of the vanadium chloroperoxidase from the fungus Curvularia inaequalis. The data show that the kinetics of this enzyme resemble that of the vanadium bromoperoxidase from the seaweed Ascophyllum nodosum. At low pH, chloride inhibited the enzyme, but the inhibition was of a dual nature. At pH 4.1 a mixed type of inhibition by chloride with respect to hydrogen peroxide was observed whereas at pH 3.1 the nature of the inhibition became competitive. The log Km for hydrogen peroxide decreased linearly with pH with a slope of -1 in the pH range 3-5. A reaction mechanism is presented to explain the observed data. We also showed that this class of enzymes is inhibited by nitrate. At pH 5.5, nitrate inhibits the chlorination reaction competitively with respect to chloride (Ki = 2 mM) and uncompetitively with respect to hydrogen peroxide. Furthermore, we showed that the enzyme produces HOCl as a reaction product. The enzyme exhibited a high thermostability (tm of 90 degrees C) and displayed high stability in organic solvents (solutions of 40% methanol, ethanol or 2-propanol) and moderate stability in the presence of the chaotropic agent guanidine/HCl (G1/2, the concentration of guanidine/HCl at which the enzyme activity was half the original activity was 3.7 M).

Chloride Peroxidase↗
PubMed1992

Defining the involvement of HOCl or Cl2 as enzyme-generated intermediates in chloroperoxidase-catalyzed reactions.

Peroxidatic substrates, catechol (CAT) and 2,4,6-trimethylphenol (TMP) were used as probes of thechloride dependent reactions catalyzed by chloroperoxidase (CPO). TMP is consumed only in the presence of chloride. TMP is a competitive inhibitor versus CAT, but CAT is a noncompetitive inhibitor versus TMP in chloride-dependent CPO-catalyzed peroxidation reactions. The ratio of TMP versus CAT consumed by the chloride-dependent CPO reaction in direct competition studies increases as the chloride concentration is increased from 1.0 to 400 mM. Ratios of non-enzymatic HOCl reactions under conditions otherwise similar to those of the CPO reactions are relatively insensitive to changes in chloride concentration and are experimentally indistinguishable from the values attained by the enzyme system at high chloride concentrations. Comparison of enzymatic ratios with those of the HOCl reactions indicate that the proportion of the enzymatic reaction involving a freely dissociable, enzyme-generated, oxidized halogen species varies from 10% at low chloride concentrations to essentially 100% at high chloride concentrations. All data are consistent with a mechanism in which chloride competes with CAT for binding to both CPO compound I and the CPO chlorinating intermediate (EOCl). Chloride binding to CPO compound I leads to the formation of EOCl and initiates the CPO chloride-dependent pathway. When CAT binds to either compound I or EOCl, it is directly oxidized to product. When chloride binds to EOCl, it either induces release of HOCl or reacts with EOCl to produce Cl2, which is released from the enzyme. TMP and CAT compete for reaction with the free oxidized halogen species. This is the first direct evidence for kinetically significant involvement of a free oxidized halogen species as an intermediate in any CPO-catalyzed reaction.

Catechols↗
PubMed2005

Prosthetic heme modification during halide ion oxidation. Demonstration of chloride oxidation by horseradish peroxidase.

Myeloperoxidase (MPO), eosinophil peroxidase (EPO), and chloroperoxidase can oxidize iodide, bromide, and chloride, but most peroxidases, including the prototypical horseradish peroxidase (HRP), reportedly only oxidize iodide and, in some cases, bromide. We report here that incubation of HRP with Br(-) and H(2)O(2) at acidic pH results in both bromination of monochlorodimedone and modification of the heme group. Mass spectrometry indicates that the heme 2- and 4-vinyl groups are modified by either replacement of a vinyl hydrogen by a bromide or addition of HOBr to give a bromohydrin. These reactions do not occur if protein-free heme and Br(-) are co-incubated with H(2)O(2) or if the HRP reaction is carried out at pH 7. Surprisingly, similar prosthetic heme modifications occur in incubations of HRP with H(2)O(2) and Cl(-). A mechanism is proposed involving oxidation of Br(-) or Cl(-) to give HOBr or HOCl, respectively, followed by addition to a vinyl group. In the reaction with Cl(-), a meso-chloro heme adduct is also formed. This first demonstration of Cl(-) oxidation by HRP, and the finding that prosthetic heme modification occurs when Br(-) or Cl(-) is oxidized in the absence of a cosubstrate, show that only modest tuning is required to achieve the unique chloride oxidation activity of MPO and EPO. The results raise the question of how the prosthetic hemes of MPO and EPO, whose function is to produce oxidized halide species, escape modification.

Bromides↗
PubMed1983

Mechanism of halide-stimulated activity of chloroperoxidase evidence for enzymatic formation of free hypohalous acid.

In acidic solutions, bromide stimulates H2O2 decomposition catalyzed by chloroperoxidase or horseradish peroxidase, altogether similar to stimulation, by chloride and bromide, of H2O2 oxidation by HOCl. Low levels of an ultra-pure NaCl inhibited chloroperoxidase catalatic activity whereas very high concentrations of this salt stimulated the reaction. The stimulation reflects low bromide contamination of the ultra-pure NaCl, which is considerably less than Br levels in most AR grade chloride salts. In all of these systems, bromide functions as a catalyst which is first oxidized to Br2 by HOX (added directly or generated enzymatically), and subsequently regenerated by H2O2 reduction of Br2. These results provide strong support for a dissociable species, most likely HOX, as the first product of chloroperoxidase catalyzed oxidation of Br- and Cl- by H2O2.

Bromates↗
PubMed1983

A steady state kinetic analysis of the reaction of chloroperoxidase with peracetic acid, chloride, and 2-chlorodimedone.

The chloroperoxidase-peracetic acid-chloride-2-chlorodimedone system is used as a model for the investigation of enzymatic halogenation reactions. Systematic variation of the concentrations of the three substrates under steady state conditions yields sets of kinetic parameters containing both kinetic and mechanistic information. Three distinct enzyme species are involved in the halogenation cycle: native enzyme, compound I, and a short-lived halogenating intermediate. Analysis of the kinetic data is complicated by the fact that chloride serves as a substrate in the second step and as an inhibitor of the first step of the overall halogenation reaction. The inhibitor binding site on the native enzyme must be protonated prior to the binding of chloride. Chloride appears to be a competitive inhibitor for both compound I formation and cyanide binding to chloroperoxidase. Only the latter reaction can be studied directly in stopped-flow experiments since compound I disappears rapidly by reacting with chloride present in solution. Rate constants are calculated for the individual steps of the reaction at four different pH values. The rate constant for compound I formation is independent of pH, but the actual rate is reduced at lower pH values due to pH dependence of the chloride inhibition. The rate constant for the oxidation of chloride by compound I decreases with increasing pH. The rate of the halogenation step appears to be independent of pH.

Acetates↗
PubMed2003

Conversion of adamsite (phenarsarzin chloride) by fungal manganese peroxidase.

Fungal manganese peroxidase was found to convert the persistent chemical warfare agent adamsite (phenarsarzin chloride) in a cell-free reaction mixture containing sodium malonate, Mn(2+) ions, and reduced glutathione. The organo-arsenical compound disappeared completely within 48 h accompanied by the formation of a more polar metabolite with a clearly modified UV spectrum. Thus, As(III) in the adamsite molecule was oxidized by manganese peroxidase to As(V) which added dioxygen and released chloride.

Arsenicals↗
PubMed1988

Purification and characterization of a novel bacterial non-heme chloroperoxidase from Pseudomonas pyrrocinia.

The first bacterial chloroperoxidase that is capable of catalyzing the chlorination of indole to 7-chloroindole was detected in Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin (Wiesner, W., van Pée, K.H., and Lingens, F. (1986) FEBS Lett. 209, 321-324). Here we describe the purification and characterization of this bacterial non-heme chloroperoxidase. The enzyme was purified by DEAE-cellulose chromatography at different pH values, molecular sieve chromatography, and Bio-Gel HTP hydroxylapatite. After the last purification step, chloroperoxidase was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. Based on gel filtration and ultracentrifugation results, the molecular weight of the enzyme was 64,000 +/- 3,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band with the mobility of a 32,000 molecular weight species. Therefore, in solution at neutral pH, this chloroperoxidase is a dimer. The enzyme did not exhibit any absorbance in the visible region of the spectrum. The isoelectric point was 4.1. Chloroperoxidase was specific for I-, Br-, and Cl- and was not inhibited by azide, but was inhibited by cyanide and F-. This procaryotic chloroperoxidase catalyzed the bromination of monochlorodimedone but not its chlorination and has no peroxidase or catalase activity. The pH optimum of the enzyme was between 4.0 and 4.5, and the enzyme was stable between pH 3.5 and 8.5 and showed no loss of activity when incubated at 60 degrees C for 2 h. Chloroperoxidase also chlorinated 4-(2-amino-3-chlorophenyl) pyrrole to yield aminopyrrolnitrin, the immediate precursor of pyrrolnitrin. This suggests very strongly that chloroperoxidase is involved in the biosynthesis of the antibiotic pyrrolnitrin.

Amino Acid Sequence↗
PubMed2001

Oxidation of bis(terpyridine)cobalt(II) chloride by cytochrome c peroxidase compounds I and II.

The bis(terpyridine)cobalt(II), Co(terpy)2(2+), reduction of cytochrome c peroxidase compound I, CcP-I, has been investigated using stopped-flow techniques as a function of ionic strength in pH 7.5 buffers at 25 degrees C. Co(terpy)2(2+) initially reduces the Trp191 radical site in CcP-I with an apparent second-order rate constant, k2, equal to 6.0+/-0.4x10(6) M(-1)s(-1) at 0.01 M ionic strength. A pseudo-first-order rate constant of 480 s(-1) was observed for the reduction of CcP-I by 79 microM Co(terpy)2(2+) at 0.01 M ionic strength. The one-electron reduction of CcP-I produces a second enzyme intermediate, CcP compound II (CcP-II), which contains an oxyferryl, Fe(IV), heme. Reduction of the Fe(IV) heme in CcP-II by Co(terpy)2(2+) shows saturation kinetics with a maximum observed rate constant, k3max, of 24+/-2 s(-1) at 0.01 M ionic strength. At low reductant concentrations, the apparent second-order rate constant for Co(terpy)2(2+) reduction of CcP-II, k3, is 1.2+/-0.5x10(6) M(-1) s-1. All three rate constants decrease with increasing ionic strength. At 0.10 M ionic strength, values of k2, k3, and k3max decrease to 6.0+/-0.8x10(5) M(-1) s(-1), 1.2+/-0.5x10(5) M(-1) s(-1), and 11+/-3 s(-1), respectively. Both the product, Co(terpy)2(3+), and ferricytochrome c inhibit the rate of Co(terpy)2(2+) reduction of CcP-I and CcP-II. Gel-filtration studies show that a minimum of two Co(terpy)2(3+) molecules bind to the native enzyme in low ionic strength buffers.

Cytochrome c Group↗
PubMed1975

Permeability of rabbit corneal epithelium to horseradish peroxidase after the influence of benzalkonium chloride.

A tight barrier against permeation of horseradish peroxidase into the corneal epithelium exists at the corneal surface adjacent to the tear film. The present light and transmission electron microscopic study reveals that the cationic surfactant, benzalkonium chloride, which is commonly added to eye drops as a preservative, breaks down this barrier. Lysis of the cell membranes was demonstrated, resulting in a leakage of the tracer into and underneath the superficial cells. The grade of cellular destruction caused by benzalkonium chloride was dependent upon the concentration and exposure time of the drug.

Animals↗
PubMed1980

N-Demethylation reactions catalyzed by chloroperoxidase.

The peroxidase-supported N-demethylations catalyzed by chloroperoxidase, a heme protein isolated from Caldariomyces fumago, have been investigated as models for cytochrome P-450-catalyzed N-dealkylations. The turnover number for the ethyl hydrogen peroxide-supported dealkylation of N,N-dimethylaniline by chloroperoxidase (1476) was much greater than that for cytochrome P-450-catalyzed dealkylations. The dealkylations of N,N-dimethylaniline by chloroperoxidase yielded N-methylaniline and formaldehyde in equimolar amounts with no other products detectable by high pressure liquid chromatography analysis of the reaction mixture. Ethyl hydrogen peroxidase could be replaced by other hydroperoxides, peroxides, or peracids. Chloride ions stimulated the reaction at low pH. The dealkylation reaction exhibited normal Michaelis-Menten saturation kinetics with respect to N,N-dimethylaniline (Km = 0.08 mM) and ethyl hydrogen peroxide (Km = 0.8 mM) at low substrate concentrations. However, substrate inhibition occurred at higher concentrations of N,N-dimethylaniline. The chloroperoxidase-catalyzed demethylations were inhibited by inhibitors of cytochrome P-450 such as azide or n-propyl gallate, but not by metyrapone, SKF-525A, or piperonyl butoxide. Although tiron and DL-epinephrine, trapping agents for the superoxide anion, inhibited the demethylation reactions, superoxide dismutase had no effect. There was no significant inhibition by alpha-phenyl-t-butyl-nitrone or 5,5-dimethyl-pyrroline-N-oxide, which react with free radicals. Diphenylfuran and DL-histidine, which react with singlet oxygen, did not inhibit the reaction. Substitution of D2O for H2O resulted in a marked inhibition with a solvent isotope effect (VH/VD) of 3.6. Chloroperoxidase did not catalyze the demethylation of N,N-dimethylaniline-N-oxide, indicating that the reaction does not proceed via an N-oxide intermediate.

Aniline Compounds↗