The kinetic properties producing the perfunctory pH profiles of catalase-peroxidases

Many structure-function relationship studies performed on the catalase-peroxidase enzymes are based on limited kinetic data. To provide a more substantive understanding of catalase-peroxidase function, we undertook a more exhaustive evaluation of catalase-peroxidase catalysis as a function of pH. Kinetic parameters across a broad pH range for the catalase and peroxidase activities of E. coli catalase peroxidase (KatG) were obtained, including the separate analysis of the oxidizing and reducing substrates of the peroxidase catalytic cycle. This investigation identified ABTS-dependent inhibition of peroxidase activity, particularly at low pH, unveiling that previously reported pH optima are clearly skewed. We show that turnover and efficiency of peroxidase activity increases with decreasing pH until the protein unfolds. The data also suggest that the catalase pH optimum is more complex than it is often assumed to be. The apparent optimum is in fact the intersection of the optimum for binding (7.00) and the optimum for activity (5.75). We also report the apparent pK(a)s for binding and catalysis of catalase activity as well as approximate values for certain peroxidatic and catalatic steps.     

Structure of azide methemoglobin

We have compared the structures of horse azide methemoglobin and methemoglobin (MetHb) at 2.8 Å resolution by X-ray difference Fourier analysis. Of four low-spin liganded Hb derivatives (nitric oxide Hb, carbon monoxide Hb, cyanide MetHb, and azide MetHb), azide MetHb is closest in structure to MetHb. In azide MetHb the ligands are co-ordinated end-on at angles of about 125 ° to the heme axes, which is similar to the stereochemistry assumed by azide in binding to free heme. Because of its bent binding geometry, azide encounters less interference in binding and perturbs the protein structure less than carbon monoxide and cyanide, which are smaller, but prefer linear axial co-ordination to heme. Steric interactions between ligand and protein are greater on the β chain, where the E helix is pushed away from the heme relative to MetHb, than on the α chain. Iron position is the same and heme stereochemistry and position are very similar in azide MetHb and MetHb.     

Retinoic acid 5,6-epoxidation by hemoproteins

Retinoic acid 5,6-epoxidase activity was found in several hemoproteins such as human oxy- and methemoglobin (HbO2 and MetHb), equine skeletal muscle oxy- and metmyoglobin (MbO2 and MetMb), bovine liver catalase, and horseradish peroxidase. Hematin also catalyzed retinoic acid 5,6-epoxidation. The results suggest that the heme moiety participates in the epoxidation. However, neither horse heart cytochrome c, nor free ferrous ion nor free ferric ion exhibited the epoxidase activity. Some hemoproteins (HbO2, MetHb, MbO2, MetMb, catalase, peroxidase, and hematin) exhibited characteristic individual pH dependences of the activity, suggesting that the epoxidase activities of the hemoproteins are influenced by the apoenzymes to some degree. This view is also supported by the finding that preincubation of an HbO2 preparation at various temperatures (37-70 degrees C) reduced its epoxidase activity with increasing temperature, whereas the activity of hematin was unaffected. Active oxygen scavengers such as mannitol, catalase, and superoxide dismutase exhibited no effect on the epoxidase activities of HbO2, MetHb, MbO2, and MetMb. A ligand of heme, CN- (100 mM), inhibited the epoxidase activities but N3- (100 mM) did not. The epoxidase activities were completely inhibited by NADPH, NADH, and/or 2-mercaptoethanol but not by NADP+ and/or NAD+. An intermediate in the epoxidation may be reduced by NADPH, NADH and/or 2-mercaptoethanol. Radical species can be considered as plausible candidates for the intermediate.     

血红蛋白对人红细胞膜流动性的影响

本文报道了pH7.5时血红蛋白和红细胞膜的结合效应.在10—45℃温度范围内观察到血红蛋白对膜脂质流动性的限制作用.看来这种限制作用不是脂质过氧化所致,而是血红蛋白和红细胞膜直接作用的结果.对流动性大的膜,血红蛋白的效应也随之增大.高铁血红蛋白及红细胞膜去胆固醇皆能修饰血红蛋白和膜的相互作用.     

The binding of cyanide to ferroperoxidase

1. The equilibrium and kinetics of cyanide binding to ferroperoxidase were investigated. At pH9.1 the equilibrium and kinetic measurements agree closely and disclose a single process with an affinity constant of 1.1x10(3)m(-1) and combination and dissociation velocity constants of 29m(-1).s(-1) and 2.5x10(-2)s(-1) respectively. 2. At pH values below 8 the affinity constant falls until at pH6.0 the ferroperoxidase.cyanide complex is no longer formed. This is shown to be associated with the formation of ferriperoxidase.cyanide complex in the mixture even in the presence of excess of sodium dithionite. 3. Rapid-pH-jump experiments show a fast pseudo-first-order interconversion between ferroperoxidase.cyanide complex at pH9.1 and ferriperoxidase.cyanide complex at pH6.0. 4. The kinetics of binding of cyanide to dithionite-reduced peroxidase at pH6.0 are complicated and radically different from those observed at pH9.1. 5. Above pH8 the change of affinity constant with pH is consistent with the undissociated species, HCN, being bound by the ferroperoxidase. The enthalpy for this process measured both by equilibrium and kinetic methods is about -8kcal/mol. 6. The binding of cyanide to reconstituted peroxidases, proto, meso and deutero, was investigated. 7. The results are discussed in relation to known data on cyanide binding to other haemoproteins.     

Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: a fluorescence quenching study on the ionic strength dependence of the interaction

The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.     

Catalytic activity and the stability of horseradish peroxidase increase as a result of its incorporation into a polyelectrolyte complex with chitosan

The incorporation of horseradish peroxidase into polyelectrolyte complexes with chitosans of different molecular weights (MW 5–150 kDa) yielded highly active and stable enzyme preparations. As a result of the selection of optimal conditions for the formation of peroxidase-chitosan complexes, it was found that 0.1% chitosan with a MW of 10 kDa had the strongest activatory effect on peroxidase (activation degree, >70%) in the reaction of o-dianisidine oxidation by hydrogen peroxide. The complex formed by 0.001% chitosan with a molecular weight of 150 kDa was most stable: when immobilized on foamed polyurethane, it retained at least 50% of the initial activity for 550 days. The highest catalytic activity was exhibited in a 0.05 M phthalate buffer (pH 5.9–6.2) by the complex containing 0.006–0.009% chitosan in the indicator reaction. The activatory effect of the polysaccharide on the enzyme was determined by its influence on the binding and conversion of the reducting substrate peroxidase.     

Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A₂ (PLA₂) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA₂ activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO₂₍₃₎ antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes.     

Cyanide binding to cytochrome c peroxidase (H52L)

Cyanide binding to a cytochrome c peroxidase (CcP) variant in which the distal histidine has been replaced by a leucine residue, CcP(H52L), has been investigated as a function of pH using spectroscopic, equilibrium, and kinetic methods. Between pH 4 and 8, the apparent equilibrium dissociation constant for the CcP(H52L)/cyanide complex varies by a factor of 60, from 135 microM at pH 4.7 to 2.2 microM at pH 8.0. The binding kinetics are biphasic, involving bimolecular association of the two reactants, followed by an isomerization of the enzyme/cyanide complex. The association rate constant could be determined up to pH 8.9 using pH-jump techniques. The association rate constant increases by almost 4 orders of magnitude over the pH range investigated, from 1.8 x 10(2) M(-1) s(-1) at pH 4 to 9.2 x 10(5) M(-1) s(-1) at pH 8.6. In contrast to wild-type CcP, where the binding of HCN is the dominant binding pathway, CcP(H52L) preferentially binds the cyanide anion. Above pH 8, cyanide binding to CcP(H52L) is faster than cyanide binding to wild-type CcP. Cyanide dissociates 4 times slower from the mutant protein although the pH dependence of the dissociation rate constant is essentially identical for CcP(H52L) and CcP. Isomerization of the CcP(H52L)/cyanide complex is observed between pH 4 and 8 and stabilizes the complex. The isomerization rate constant has a similar magnitude and pH dependence as the cyanide dissociation rate constant, and the two reactions are coupled at low cyanide concentrations. This isomerization has no counterpart in the wild-type CcP/cyanide complex.     

Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase

Stable polyelectrolyte capsules were produced by the layer-by-layer (LbL) assembling of biodegradable polyelectrolytes, dextran sulfate and protamine, on melamine formaldehyde (MF) microcores followed by the cores decomposition at low pH. The mean diameter of the capsules at pH 3-5 was 8.0 +/- 0.2 microm, which is more than that diameter of the templates (5.12 +/- 0.15 microm). With pH growing up to 7-8, the capsules enlarged, swelling up to the diameter 9-10 microm. The microcapsules were loaded with horseradish peroxidase. Seemingly, peroxidase is embedded in the gellike structure in the microcapsule interior formed by MF residues in the complex with polymers used for LbL coating as proved by Raman confocal spectroscopy. The amount of finally incorporated peroxidase increased from 0.2 x 10(8) to 2.2 x 10(8) peroxidase molecules per capsule with pH growing from 5 to 8. The pH shifts causing changes in capsule swelling and the replacement of solutions without pH shifts lead to the protein loss. The encapsulated peroxidase showed a high activity (57%), which remained stable for 12 months.     

Covalent binding of peroxidase to CH- and AH-Sepharose 4 B]

The properties of peroxidase insolubilized by covalent binding to CH- and AH-Sepharose 4 B in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are described. CH-Sepharose 4 B bound peroxidase yields an enzyme preparation with a residual specific activity of 60.6%. When bound to AH-Sepharose 4 B, the residual specific activity is to 78%. The reasons of these differences in the catalytic activity of the two insolubilized enzyme preparations are discussed. By covalent binding on CH- and AH-Sepharose 4 B, peroxidase exibits no changes in its pH optimum; it virtually keeps the same activity after being used ten times. Insolubilized peroxidase preparations, dried and reimbibed after being stored for 6 weeks at room temperature still display 50% of the initial specific activity of the insolubilized enzyme. Stored in acetate buffer, the enzyme preparations maintain their activity during all this interval.     

Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase.

Peroxidases typically bind their reducing substrates weakly, with K(d) values in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferric horseradish peroxidase isoenzyme C (HRPC) [K(d) = 2.4 microM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provided a useful tool for probing the environment of the peroxidase aromatic-donor-binding site and the distal heme cavity. Knowledge of the underlying thermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isothermal titration calorimetry results of this study on the binding of aromatic hydroxamic acid analogues to ferric HRPC under nonturnover conditions (no H(2)O(2) present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to HRPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. The overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, with the distal Arg38 acting as an "entropic gate keeper". Dramatic enthalpy-entropy compensation is observed for BHA and 2-naphthohydroxamic acid binding to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due to increased flexibility of Arg38 in the complexes at low pH and greater access by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the K(d) values corrected for substrate ionization suggests that enthalpy-entropy compensation is operative over a wide pH range. Examination of the thermodynamics of binding of ring-substituted hyrazides to HRPC reveals that the binding affinities of aromatic donors are highly sensitive to the position and nature of the ring substituent.     

Interaction of Gold(I) with the Active Site of Selenium-Glutathione Peroxidase

Gold(I) thioglucose in the presence of excess glutathione (GSH) leads to strong and reversible inhibition of selenium-glutathione peroxidase (EC 1.11.19) around neutral pH. Binding at equilibrium and competition studies demonstrated that the most reduced form of the active site selenocysteine is the only binding site for gold(I) Steady-state kinetics indicate that gold(I) forms a dead-end complex with glutathione peroxidase in competition with the reduction of hydroperoxide. The apparent K₁ is 2 3 μM at pH 7.6,37°C and 1 mM GSH. Theoretical models of inhibition were assessed by the use of linear least-squares fitting to a generalized integrated rate equation. The results are consistent with trapping of gold(I) at the active site in the form of a mixed bidentate selenolato-thiolate complex involving GSH and the active site selenocysteine. The kinetics of inhibition imply that the resting form of glutathione peroxidase in the presence of excess GSH is also within the enzyme cycle. This rules out the existence of selenium(+lV) species in the redox cycle of the active site when t-butylhydroperoxide is used as a substrate. Electronic properties of selenium and gold as well as a large relief of inhibition by selenocysteine suggest that a very stable interaction should be obtained between Se(-II) and gold(I) through covalent bonding. These results suggest that glutathione peroxidase could be a target of gold drugs used in the treatment of rheumatoid arthritis.     

Superoxide dismutase activity and methemoglobin content of human and animal erythrocytes

In healthy newborn babies, superoxide dismutase activity and MetHb content of the erythrocytes are higher than those in adult subjects. It was also demonstrated that low activity of superoxide dismutase in guinea pigs, albino mice and teleost fish Coregonus autumnalis (in autumn period) is paralleled by a higher level of MetHb. On the contrary, high enzymic activity in albino rats and C. autumnalis (spring period) accounts of a lower level of MetHb. Seasonal changes in the activity of superoxide dismutase were found in guinea pigs and fish.     

Kinetics of iron and copper catalysis of ascorbate oxidation.

The effects of oxidant, pH and ligands on iron- and copper-catalyzed ascorbate oxidation have been examined. The formation of the catalyst-substrate complex is affected by pH, whereas oxidant affects its breakdown. With copper-ion catalysis, ligands inhibit competitively. With iron catalysis, on the other hand, for a series of aminopolycarboxylic ligands at neutral pH, formation of catalyst-substrate is favored by ligands which form more stable iron complexes. Decreased rates caused by changes in metal environment (ligand or pH) may result for competing activities (e.g., catalase activity competing with peroxidase activity). Evidence for a ternary complex (catalyst-substrate-oxidant) is presented.     

Lignin peroxidase of Phanerochaete chrysosporium. Evidence for an acidic ionization controlling activity

The active site amino acid residues of lignin peroxidase are homologous to those of other peroxidases; however, in contrast to other peroxidases, no pH dependence is observed for the reaction of ferric lignin peroxidase with H2O2 to form compound I (Andrawis, A., Johnson, K.A., and Tien, M. (1988) J. Biol. Chem. 263, 1195-1198). Chloride binding is used in the present study to investigate this reaction further. Chloride binds to lignin peroxidase at the same site as cyanide and hydrogen peroxide. This is indicated by the following. 1) Chloride competes with cyanide in binding to lignin peroxidase. 2) Chloride is a competitive inhibitor of lignin peroxidase with respect to H2O2. The inhibition constant (Ki) is equal to the dissociation constant (Kd) of chloride at all pH values studied. Chloride binding is pH dependent: chloride binds only to the protonated form of lignin peroxidase. Transient-state kinetic studies demonstrate that chloride inhibits lignin peroxidase compound I formation in a pH-dependent manner with maximum inhibition at low pH. An apparent pKa was calculated at each chloride concentration; the pKa increased as the chloride concentration increased. Extrapolation to zero chloride concentration allowed us to estimate the intrinsic pKa for the ionization in the lignin peroxidase active site. The results reported here provide evidence that an acidic ionizable group (pKa approximately 1) at the active site controls both lignin peroxidase compound I formation and chloride binding. We propose that the mechanism for lignin peroxidase compound I formation is similar to that of other peroxidases in that it requires the deprotonated form of an ionizable group near the active site.     

pH-dependent binding of immunoglobulins to intestinal cells of the neonatal rat

Rat and rabbit IgG immunoglobulins conjugated to horseradiah peroxidase as a histochemical marker bind at 0 degrees C to the luminal surface of absorptive cells in isolated segments of jejunum from 10-12-day old rats. Binding is observed at pH 6.0, near the normal luminal pH of the duodenum and jejunum at this age, but not at pH 7.4. Furthermore, no binding occurs when cells are exposed at pH 6.0 to either free peroxidase or peroxidase conjugated to chicken or sheep IgG immunoglobulins or bovine serum albumin. The sensitivity of binding to pH suggests a means whereby immunoglobulins which are selectively absorbed by the cells can be released efficiently at the abluminal surface.     

Stepwise binding of nickel to horseradish peroxidase and inhibition of the enzymatic activity

The incubation of horseradish peroxidase C (HRPC) with millimolar concentrations of nickel, at room temperature and at pH 4.0, induced the progressive formation of a metal-enzyme complex characterized by alterations of the enzyme Soret absorption band that were time- as well as nickel concentration- dependent. For any given incubation period between 1 and 60 min, 2 values for the apparent dissociation constant (K(d)) were found, suggesting the presence of binding sites with different affinities for nickel. The value of each K(d) dropped as the incubation time increased, indicating a progressive stabilization of the metal-enzyme complex. Hill plots suggested a cooperative binding of up to four Ni2+ ions per molecule of HRPC. The inhibition of the enzymatic activity by nickel was studied by following the H2O2-mediated oxidation of o-dianisidine by HRPC under steady-state kinetic conditions. Ni2+ was found to be either a noncompetitive or a mixed inhibitor of HRPC depending both on the duration of preincubation with the enzyme and on Ni2+ concentration. The enzyme remained active only over a limited metal concentration range and data indicated that binding of one Ni2+ affected the substrate binding site, binding of a second Ni2+ affected both substrate and peroxide binding sites, and binding of more than 2 Ni2+ per HRPC molecule led to complete loss of enzymatic activity. Results pointed to the damaging effects of prolonged exposure to heavy metals and also to the existence of a critical metal concentration beyond which immediate abolishing of enzymatic activity was observed.     

Cationic ascorbate peroxidase isoenzyme II from tea: structural insights into the heme pocket of a unique hybrid peroxidase

The novel class III ascorbate peroxidase isoenzyme II from tea leaves (TcAPXII), with an unusually high specific ascorbate peroxidase activity associated with stress response, has been characterized by resonance Raman (RR), electronic absorption, and Fourier transform infrared (FT-IR) spectroscopies. Ferric and ferrous forms and the complexes with fluoride, cyanide, and CO have been studied at various pH values. The overall blue shift of the electronic absorption spectrum, the high RR frequencies of the core size marker bands, similar to those of 6-coordinate low-spin heme, and the complex RR spectrum in the low-frequency region of ferric TcAPXII indicate that this protein contains an unusual 5-coordinate quantum mechanically mixed-spin heme. The spectra of both the fluoride and the CO adducts suggest that these exogenous ligands are strongly hydrogen-bonded with a residue that appears to be unique to this peroxidase. Electronic absorption spectra also emphasize structural differences between the benzhydroxamic acid binding sites of TcAPXII and horseradish peroxidases (HRPC). It is concluded that TcAPXII is a paradigm peroxidase since it is the first example of a hybrid enzyme that combines spectroscopic signatures, structural elements, and substrate specificities previously reported only for distinct class I and class III peroxidases.     

Complex formation between the copper protein, azurin and the cytochrome c peroxidase of Pseudomonas aeruginosa.

Reduced azurin reacts with the resting, oxidized cytochrome c peroxidase of Pseudomonas aeruginosa to yield time courses observed at 420 nm, which consist of the sum of two exponential processes. Each process exhibits a hyperbolic dependence of the observed rate constant on the reduced azurin concentration. The fraction of the total optical density change which each process contributes is found to be dependent on the reduced azurin concentration. This pattern of reactivity is maintained at pH values between 5.5 and 8.0. The data has been analyzed in terms of a complex formation between the two proteins followed by an intramolecular electron exchange reaction. This analysis yields values for the binding constants at each pH value. The intramolecular exchange reaction is independent of pH, whilst the pH dependence of the binding reaction suggests the involvement of a histidine residue in this process.