The VI international conference on prostaglandins and related compounds

The effects of a heme ligand, cyanide, on pure ovine prostaglandin H synthase have been examined in detail as one approach to elucidating the role of the heme cofactor in cyclooxygenase and peroxidase catalysis by the synthase. Cyanide bound to the synthase heme with an affinity (Kd) of 0.19 mM, and inhibited the peroxidase activity of the synthase, with a K_I value of 0.23 mM. Cyanide increased the sensitivity of the cyclooxygenase to inhibition by the peroxide scavenger, glutathione peroxidase. This increased sensitivity to inhibition reflect and increase in the level of peroxide required to activate the cyclooxygenase, from 21 nM in absence of cyanide to over 300 nM when 2.5 mM cyanide was present. The increase in peroxide activator requirement with increasing cyanide concentration closely paralleled the formation of the holoenzyme-cyanide complex. These effects of low levels of cyanide suggest that the heme prosthetic group of the synthase participates in the efficient activation of the cyclooxygenase by peroxide. Cyanide blocked the stimulation of cyclooxygenase velocity by phenol, but not the phenol-induced increase in overall oxygen consumption. This blockade by cyanide was noncompetitive with respect to phenol and was characterized by a K_I of 4 mM. The higher K_I value for this effect suggests that cyanide can also interact at a site other than the heme prosthetic group. The role of the heme prosthetic group in promoting efficient activation of the cyclooxygenase by peroxide appears to be central to the ability of the synthase to amplify the ambient peroxide concentration rapidly.     

Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis

High levels of NaCN (20 to 250 mM) were required to inhibit cyclooxygenase catalysis and cause extended lag periods (up to 1.6 min), whereas CO failed to inhibit catalysis. This NaCN inhibition was easily overcome by endogenous or exogenous hydroperoxides. Added hydroperoxides acted to eliminate lag periods without undergoing net conversion to other chemical species. In addition, experiments with glutathione peroxidase inhibition showed that hydroperoxides were essential not only in the early phases, but throughout catalysis. In spectrophotometric experiments, NaCN formed a complex with ferriheme cyclooxygenase (Kd = 1.3 mM) and inhibited hydroperoxide interaction with this form of the enzyme. Phenolic antioxidants, only slightly extended lag periods while inhibiting oxygenation rates more than 50%. Low levels of phenol (which is normally stimulatory) or alpha-naphthol when combined with NaCN or glutathione peroxidase (agents which interfere with peroxide activation) resulted in potent synergistic inhibition with long lag times. A mechanism consistent with all of the above properties of cyclooxygenase has been elucidated, Further mechanistic explanation was sought for reaction-catalyzed self-inactivation of cyclooxygenase. This phenomenon could not be explained simply by heme lability, or cyclooxygenase sensitivity to destruction by ambient hydroperoxides, Rather, it appears to involve a destructive reaction intermediate intrinsic to involve a destructive reaction intermediate intrinsic to the cyclooxygenase mechanism.     

Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities

The peroxidase activity of prostaglandin H (PGH) synthase catalyzes reduction of 5-phenyl-4-pentenyl hydroperoxide to 5-phenyl-4-pentenyl alcohol with a turnover number of approximately 8000 mol of 5-phenyl-4-pentenyl hydroperoxide/mol of enzyme/min. The kinetics and products of reaction establish PGH synthase as a classical heme peroxidase with catalytic efficiency similar to horseradish peroxidase. This suggests that the protein of PGH synthase evolved to facilitate peroxide heterolysis by the heme prosthetic group. Comparison of an extensive series of phenols, aromatic amines, beta-dicarbonyls, naturally occurring compounds, and nonsteroidal anti-inflammatory drugs indicates that considerable differences exist in their ability to act as reducing substrates. No correlation is observed between the ability of compounds to support peroxidatic hydroperoxide reduction and to inhibit cyclooxygenase. In addition, the resolved enantiomers of MK-410 and etodolac exhibit dramatic enantiospecific differences in their ability to inhibit cyclooxygenase but are equally potent as peroxidase-reducing substrates. This suggests that there are significant differences in the orientation of compounds at cyclooxygenase inhibitory sites and the peroxidase oxidation site(s). Comparison of 5-phenyl-4-pentenyl hydroperoxide reduction by PGH synthase and horseradish peroxidase reveals considerable differences in reducing substrate specificity. Both the cyclooxygenase and peroxidase activities of PGH synthase inactivate in the presence of low micromolar amounts of hydroperoxides and arachidonic acid. PGH synthase was most sensitive to arachidonic acid, which exhibited an I50 of 0.6 microM in the absence of all protective agents. Inactivation by hydroperoxides requires peroxidase turnover and can be prevented by reducing substrates. The I50 values for inactivation by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid are 4.0 and 92 microM, respectively, in the absence and presence of 500 microM phenol, a moderately good reducing substrate. The ability of compounds to protect against hydroperoxide-induced inactivation correlates directly with their ability to act as reducing substrates. Hydroquinone, an excellent reducing substrate, protected against hydroperoxide-induced inactivation when present in less than 3-fold molar excess over hydroperoxide. The presence of a highly efficient hydroperoxide-reducing activity appears absolutely essential for protection of the cyclooxygenase capacity of PGH synthase. The peroxidase activity is, therefore, a twin-edged sword, responsible for and protective against hydroperoxide-dependent inactivation of PGH synthase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical modification of prostaglandin endoperoxide synthase by N-acetylimidazole. Effect on enzymic activities and EPR spectroscopic properties.

Prostaglandin H synthase apoprotein, without its prosthetic heme group, was inactivated by N-acetylimidazole under conditions typical for the O-acetylation of tyrosyl residues. A spontaneous reactivation occurred above pH 7.5 at 22 degrees C, which indicated spontaneous hydrolysis of acetylated residues. Below pH 7.5, where stable inactivation was observed, reactivation was achieved by reaction with hydroxylamine. Both enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, were inactivated and reactivated simultaneously and to the same extent. In contrast to the apoprotein, the holoenzyme with heme was not inactivated by N-acetylimidazole. The number of acetyl groups, as determined as hydroxamate after the reaction with hydroxylamine at pH 8.2, was 2.5 +/- 0.4 for the apoprotein and 1.0 +/- 0.24 for the holoenzyme. The specific binding of heme as the prosthetic group was no longer observed by EPR (signals at g = 6.7 and 5.3) when hemin was added to the N-acetylimidazole-reacted apoprotein. Treatment of N-acetylimidazole-reacted apoprotein with hydroxylamine restored the specific binding of heme. The N-acetylimidazole-reacted apoprotein supplemented with hemin and reacted with hydroperoxides, neither showed electronic absorption spectra of higher oxidation states nor an EPR doublet signal due to a tyrosyl radical. These results demonstrate that heme protects against the inactivating modification by N-acetylimidazole and that this modification prevents binding of the prosthetic heme group necessary for both enzymic activities. The absence of the prosthetic heme group explains the concomitant loss of cyclooxygenase and peroxidase activities, as well as the absence of higher oxidation states and the tyrosyl radical. We suggest that the acetylation of a residue in the heme pocket, most probably a tyrosine, although a histidine cannot be definitely disproved, exerts the inhibiting effects. This residue could be the axial ligand of the heme or in close contact to the heme. The results also show that the inhibition by N-acetylimidazole does not involve the acetylation of Ser530 which causes the inhibition by acetylsalicylic acid of cyclooxygenase. [The numbering of amino acids in ovine prostaglandin H synthase is according to DeWitt, D. L. and Smith, W. L. (1988) Proc. Natl Acad. Sci. USA 85, 1412-1416 including a signal peptide of 24 residues which is missing in the processed protein.     

Cytochrome c peroxidase activity of bovine heart cytochrome oxidase incorporated in liposomes and generation of membrane potential

Cytochrome oxidase vesicles catalyzed the peroxidatic oxidation of ferrocytochrome c. The maximal peroxidase activity in the absence of an uncoupling agent was 9.8 mol ferrocytochrome c oxidized/(s X mol heme a), indicating a 5-fold activation compared with the soluble enzyme system. The peroxidase activity was further enhanced 1.2 to 2.1 times upon addition of an uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The stoichiometry of the reduction of hydrogen peroxide by ferrocytochrome c was established to be 1 : 2, indicating water formation. Potassium cyanide (0.14 mM) completely inhibited the peroxidase activity. The inhibition by 1 mM CO was 40-77% depending on the energized state of cytochrome oxidase vesicles, but in contrast, 85% inhibition was observed with the soluble enzyme. In the energized state the enzyme showed a slightly lower affinity for CO than in the deenergized state. Coupled with the peroxidase activity, a membrane potential of 72 mV was registered transiently; this may be physiologically significant in relation to the energy transduction mechanism.     

Similarities in the optical spectra of prostaglandin H synthase during its cyclooxygenase and peroxidase reactions

The spectral behavior of the enzyme prostaglandin H synthase was studied in the Soret region under conditions that permitted comparison of enzyme intermediates involved in peroxidase and cyclooxygenase activities. First, the peroxidase activity was examined. The enzyme's spectral behavior upon reacting with 5-phenyl-pent-4-enyl-1-hydroperoxide was different depending on the presence or absence of the reducing substrate, phenol. In the reaction of prostaglandin H synthase with the peroxide in the absence of phenol, formation of the enzyme intermediate compound I is observed followed by partial conversion to compound II and then by enzyme bleaching. In the reaction with both peroxide and phenol the absorbance decreases and a steady-state spectrum is observed which is a mixture of native enzyme and compound II. The steady state is followed by an increase in absorbance back to that of the native enzyme with no bleaching. The difference can be explained by the reactivity of phenol as a reducing substrate with the prostaglandin H synthase intermediate compounds. Cyclooxygenase activity with arachidonic acid could not be examined in the absence of diethyldithiocarbamate because extensive bleaching occurred. In the presence of diethyldithiocarbamate, enzyme spectral behavior similar to that seen in the reaction of the peroxide and phenol was observed. The similarity of the spectra strongly suggests that the enzyme intermediates involved in both the peroxidase and cyclooxygenase reactions are the same.     

Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.     

Thromboxane A2 synthase. Modification during "suicide" inactivation.

Thromboxane synthase is a ferrihemoprotein which undergoes mechanism-based inactivation during catalysis. This "suicide" process may be an important factor for limiting thromboxane A2 biosynthesis in cells. Although the kinetics have been characterized for purified enzyme and platelets, the chemical basis for inactivation has remained unclear. Protein modification or alteration of the heme prosthetic group is each compatible with the irreversible nature of suicide inactivation of thromboxane synthase. We have investigated these two possibilities using enzyme purified to homogeneity. Our data show that the Soret absorbance spectrum of thromboxane synthase is unaltered by additions of prostaglandin endoperoxide H2 which cause enzymatic inactivation. Using a coupled cyclooxygenase/thromboxane synthase system and polyacrylamide gel electrophoresis we have demonstrated that the enzyme retains radiolabel under nondenaturing gel conditions. Label incorporation is reduced by the competitive thromboxane synthase inhibitor U63557, an agent that also protects the enzyme from inactivation. Under denaturing conditions the radiolabel localizes with the released heme prosthetic group. In addition, interaction of the heme prosthetic group with cyanide was prevented by inactivating the enzyme with prostaglandin H2. In similar experiments, the lipid hydroperoxide 15(S)-hydroperoxyeicosatetraenoic acid inactivated thromboxane synthase with concurrent bleaching of the Soret spectrum. Labeling studies with a coupled soybean lipoxygenase/thromboxane synthase system indicate that, in this case, the apoenzyme is modified. These results suggest that the mechanism of thromboxane synthase inactivation during thromboxane A2 biosynthesis involves a tight, nondestructive association of substrate or product with the prosthetic heme group. Inactivation by hydroperoxides, however, appears to result from apoenzyme modification. These reactions may have important implications for cellular physiology and pathophysiology of thrombosis.     

Characterization of hog thyroid peroxidase

Several fundamental properties of purified hog thyroid peroxidase (A413 nm/A280 nm = 0.55) were investigated in comparison with bovine lactoperoxidase. The Mr of thyroid peroxidase was 71,000. The prosthetic group of thyroid peroxidase was identified spectrophotometrically as protoheme IX after the enzyme was hydrolyzed with Pronase. Optical spectra of oxidized and reduced thyroid peroxidases and their complexes with azide and cyanide were very similar to lactoperoxidase, except that lactoperoxidase had two reduced forms with the Soret band either at 446 or 435 nm, and thyroid peroxidase lacked a reduced form having the 446-nm band. From comparison of their pyridine hemochrome spectra, epsilon mM at 413 nm of thyroid peroxidase was estimated to be 114, being the same as that of lactoperoxidase. The cyanide inhibition for the reaction of thyroid peroxidase was competitive with hydrogen peroxide and the inhibition constant was in rough accord with the dissociation constant of its cyanide complex measured from spectrophotometric titration. Azide inhibited the reaction with an inhibition constant which was about one one-thousandth of the dissociation constant for its spectrally discernible complex. The azide inhibition was not competitive with hydrogen peroxide and decreased as the reaction proceeded. Aminotriazole inhibited the reaction strongly, and the inhibition was augmented during the reaction. These inhibition patterns of azide and aminotriazole were more or less observed in the reaction of lactoperoxidase, but not in the case of horseradish peroxidase. Characteristics of animal peroxidases are discussed.     

Anion binding to resting and half-reduced Pseudomonas cytochrome c peroxidase

The anion-binding characteristics of resting and half-reduced Pseudomonas cytochrome c peroxidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (R?nnberg, M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197-204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the gz values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 microM, 2.5 mM and 0.13 M, respectively. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.     

Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region

Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these agents with the synthase that produced inhibition of the cyclooxygenase led to a decreased accessibility of the Arg253 region to proteases. Aspirin treatment made the synthase less resistant to trypsin; aspirin-treated synthase became more resistant to trypsin when it was incubated with indomethacin before addition of the protease. The presence of 50 microM arachidonate during digestion of apoenzyme or aspirin-treated apoenzyme with trypsin did not decrease the cleavage of the synthase subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX

Catalytic turnover of sodium azide by horseradish peroxidase, which produces the azidyl radical, results in inactivation of the enzyme with KI = 1.47 mM and kinact = 0.69 min-1. Inactivation of 80% of the enzyme requires approximately 60 equiv each of NaN3 and H2O2. The enzyme is completely inactivated by higher concentrations of these two agents. meso-Azidoheme as well as some residual heme are obtained when the prosthetic group of the partially inactivated enzyme is isolated and characterized. Reconstitution of horseradish peroxidase with meso-azidoheme yields an enzyme without detectable catalytic activity even though reconstitution with heme itself gives fully active enzyme. The finding that catalytically generated nitrogen radicals add to the meso carbon of heme shows that biological meso additions are not restricted to carbon radicals. The analogous addition of oxygen radicals may trigger the normal and/or pathological degradation of heme.     

Protection of cyclooxygenase activity during heme-induced destabilization

Sheep vesicular gland cyclooxygenase is destroyed spontaneously when incubated with only substoichiometric amounts of heme. Peroxides may participate in this destruction, since glutathione peroxidase, catalase, and phenol, a cosubstrate for prostaglandin hydroperoxidase, all protect the cyclooxygenase activity. Stoichiometric or greater levels of heme also tend to protect the enzyme from inactivation. Therefore, to achieve optimal recoveries of enzyme activity during purification and storage, the addition of prostaglandin hydroperoxidase cosubstrate, such as phenol, in combination with high levels of heme is recommended. The current understanding of destabilization and protection of cyclooxygenase now allows an interpretation of the previously unexplained phenomenon of slow phenol activation of cyclooxygenase acetone powder preparations. Phenol appears to protect enzyme activity during the slow equilibration of apoenzyme with endogenous heme to form the active holoenzyme. In the absence of phenol, the progressive rise in activity is not seen as the enzyme is vulnerable to heme-induced destruction.     

Chemical modification of prostaglandin H synthase with diethyl pyrocarbonate.

The role of histidine in catalysis by prostaglandin H synthase has been investigated using chemical modification with diethyl pyrocarbonate (DEPC), an agent that has been found to rather selectively derivatize histidine residues in proteins under mild conditions. Incubation of the synthase apoprotein with DEPC at pH 7.2 resulted in a progressive loss of the capacity for both cyclooxygenase and peroxidase catalytic activities. The kinetics of inactivation of the cyclooxygenase activity were dependent on the concentration of DEPC; a second-order rate constant of 680 M-1 min-1 was estimated for reaction of the apoenzyme at pH 7.2 and 0 degrees C. The kinetics of inactivation of the cyclooxygenase by DEPC exhibited a sigmoidal dependence on the pH, indicating that deprotonation of a group with a pKa of 6.3 was required for inactivation. The presence of the heme prosthetic group slowed, but did not prevent, inactivation by DEPC. The stoichiometry of histidine modification of apoenzyme during inactivation determined from absorbance increases at 242 nm agreed well with the overall stoichiometry of derivatized residues determined with [14C]DEPC, indicating that modification by DEPC was quite selective for histidine residues on the synthase. Although modification of several histidine residues by DEPC was observed, only one of the histidine residues was essential for cyclooxygenase activity. Modification of the holoenzyme with DEPC altered the EPR signal of the hydroperoxide-induced tyrosyl free radical from the wide doublet (35 G, peak-to-trough) found with the native synthase to a narrower singlet (28 G, peak-to-trough) quite like that found in the indomethacin-synthase complex. Reaction of the indomethacin-synthase complex with DEPC was found to increase the cyclooxygenase velocity by 9 times its initial value, to about one-third of the uninhibited value, without displacement of the indomethacin; the peroxidase was significantly inactivated under the same conditions. Histidyl residues in the synthase are thus likely to have important roles not only in cyclooxygenase and peroxidase catalysis but also in the interaction of the synthase with indomethacin.     

Functional differentiation of cyclooxygenase and peroxidase activities of prostaglandin synthase by trypsin treatment. Possible location of a prosthetic heme binding site

Treatment of prostaglandin (PG)H synthase purified from ram seminal vesicle microsomes with trypsin cleaves the 70-kDa subunits into 33- and 38-kDa fragments (Chen, Y.-N. P., Bienkowski, M. J., and Marnett, L. J. (1987) J. Biol. Chem. 262, 16892-16899). In contrast to a minimal decrease in cyclooxygenase activity, peroxidase activity declines rapidly following trypsin treatment. The time course for loss of guaiacol peroxidase activity corresponds closely to the time course for protein cleavage. The ability of trypsin-treated enzyme to support catalytic reduction of 5-phenyl-4-pentenyl-1-hydroperoxide in the presence of reducing substrates is significantly reduced. The products of metabolism of 10-hydroperoxy-8,12-octadecadienoic acid indicate that trypsin-treated enzyme catalyzes homolytic scission of the hydroperoxide bond in contrast to the heterolytic scission catalyzed by intact enzyme. Spectrophotometric titrations of hematin addition to trypsin-treated PGH synthase indicate approximately a 50% reduction in heme binding. These observations suggest that trypsin treatment of PGH synthase decreases the ability of the protein to bind prosthetic heme at a site that controls peroxidase activity. Comparison of the N-terminal sequence of the 38-kDa fragment of trypsin-treated PGH synthase to the amino acid sequence of the intact protein indicates that cleavage occurs between Arg253 and Gly254. Based on literature precedents and the results of the present investigations, we propose that the heme prosthetic group that controls the peroxidase activity of PGH synthase binds to the His residue of the sequence His250-Tyr251-Pro252-Arg253 located immediately adjacent to the trypsin cleavage site.     

Prosthetic group content and ligand binding properties of a spinach catalase

A recently discovered form of spinach catalase that contains both a novel heme and protoheme as prosthetic groups has been characterized using immunological and spectroscopic techniques. The enzyme appears to be a dimer of identical Mr 60,000 monomers. Extraction of the non-covalently bound prosthetic groups, followed by thin-layer chromatography of the extract, suggested that the novel heme contains four carboxylic acid side-chain groups. The resonance Raman spectrum of the resting enzyme indicates that the protoheme prosthetic group is five-coordinate and high-spin. The enzyme was shown to bind formate, azide and cyanide. Cyanide and azide binding to catalase are biphasic, suggesting the existence of two different binding sites for cyanide and azide in the enzyme. Results obtained from EPR and resonance Raman spectroscopies also support the hypothesis that two different ligand-binding sites are present in the enzyme. Western blots suggest that the Mr 60,000 peptide of the novel heme-containing catalase is similar or identical to that of a previously characterized, exclusively protoheme-containing, tetrameric catalase.     

Prostaglandin H synthase: Resolved and unresolved mechanistic issues

The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain radical mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the H-abstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.     

Selective destruction and removal of heme from prostaglandin H synthase

Treatment of the holoenzyme form of prostaglandin H synthase with oxygen gas in the presence of excess dithionite has been found to selectively oxidize the enzyme's heme cofactor. Both the cyclooxygenase and peroxidase activities of the PGH synthase were restored upon addition of hematin. A convenient procedure has been developed to prepare milligram amounts of apo-PGH synthase from the holoenzyme. This procedure appears to involve a reactive species generated during cooxidation of dithionite and heme. The reactive species differs from that generated during the cyclooxgenase catalytic reactions which inactivates the enzyme. The heme in hemoglobin and hematin is destroyed by the same treatment. Direct addition of hydrogen peroxide converted holo-PGH synthase to the apoenzyme, but with extensive loss of enzymatic activity.     

The oxidation and decarboxylation of retinoic acid by horseradish peroxidase.

The decarboxylation of retinoic acid by horseradish peroxidase was investigated. A marked increase in the yield of products was obtained. However, the data indicated the reaction was a nonenzymatic, heme catalyzed peroxidation. Previously reported requirements for phosphate, oxygen and ferrous ion were eliminated when hydrogen peroxide was provided. Peroxide also eliminated the EDTA and cyanide induced inhibition of the phosphate dependent system. In the presence of hydrogen peroxide, horseradish peroxidase was not essential to the reaction; heme equivalent amounts of hemoglobin decarboxylated retinoic acid with equal facility. However, hemoglobin was ineffective in the absence of hydrogen peroxide. Attainment of 50--60% decarboxylation represented complete utilization of the available retinoic acid. Thus the products of the reaction can be divided into two groups, products of retinoic acid oxidation and products of an oxidative decarboxylation of retinoic acid.     

Structural Change and Catalytic Activity of Horseradish Peroxidase in Oxidative Polymerization of Phenol

The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.