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PubMed2001

Influence of brain catalase on ethanol-induced loss of righting reflex in mice.

The effect of lead acetate and 3-amino-1, 2, 4-triazole (AT) on ethanol-induced loss of righting reflex (LORR) and brain catalase activity was studied in an attempt to confirm earlier observations on the involvement of catalase in ethanol-induced effects. Lead acetate (0 or 100 mg/kg) or AT (0 or 500 mg/kg) was injected (acutely) into mice 7 days or 5 h before testing. Other mice were exposed to drinking fluid containing 500 ppm lead acetate for 60 days. On the test day, mice received an intraperitoneal injection of ethanol (4.0 or 4.5 g/kg) and the duration of LORR was recorded. Acute lead-treated animals demonstrated a reduction in the duration of the LORR. However, both chronic administration of lead acetate and AT treatment increased the duration of ethanol-produced LORR. Furthermore, brain catalase activity in acute lead pretreated animals showed a significant induction, whereas it was reduced in chronic lead and AT treated mice. These results suggest that brain catalase activity, and by implication centrally formed acetaldehyde, may modulate ethanol-induced LORR.

Animals↗
PubMed2003

Localisation of Helicobacter pylori catalase in both the periplasm and cytoplasm, and its dependence on the twin-arginine target protein, KapA, for activity.

Helicobacter pylori induces a severe inflammatory response in the gastric mucosa. It is able to withstand the inflammatory response by producing proteins such as KatA and KapA. The C-terminus of KatA possesses a unique tetra-lysine motif not found in other catalases or other known protein sequences. Mutants deficient in this motif were constructed by site-directed mutagenesis. Cytoplasmic and periplasmic catalase activities were measured for the parental strain, a truncated KatA mutant (deficient in the unique C-terminal tetra-lysine motif) and a previously constructed KapA-deficient mutant (confirming previous observations regarding the possible periplasmic localisation of KatA). No differences were observed in the cytoplasmic catalase activities, however, the KapA-deficient mutant had approximately 5.5 times less catalase activity in the periplasmic extract when compared to the periplasmic preparations of either parental strain or KatA truncated mutant. N-terminal sequencing of KatA revealed no cleaved N-terminal signal peptide, indicating Sec-independent transport. These findings support previous reports that there is some form of interaction between KatA and KapA of H. pylori, an interaction which still needs to be characterised.

Amino Acid Sequence↗
PubMedDate not supplied

Methanol metabolism and embryotoxicity in rat and mouse conceptuses: comparisons of alcohol dehydrogenase (ADH1), formaldehyde dehydrogenase (ADH3), and catalase.

Mouse embryos are more sensitive than rat embryos in response to methanol (CH(3)OH) and its ability to elicit developmental abnormalities. Intrinsic differences in the metabolism of CH(3)OH to formaldehyde (HCHO) and formic acid (HCOOH) by the enzymes alcohol dehydrogenase (ADH1), formaldehyde dehydrogenase (ADH3), and catalase may contribute to the observed species sensitivity. Specific activities for enzymes involved in CH(3)OH metabolism were determined in rat and mouse conceptuses during the organogenesis period of 8-25 somites. Spatial activity relationships were also compared separately in heads, hearts, trunks, and the visceral yolk sac (VYS) from early (7-12 somites) and late (20-22 somites) organogenesis-stage rat and mouse embryos. Catalase activities were similar between rat and mouse conceptuses. In the mouse heart, catalase activities were consistently lower when compared to other tissues. Specific activities for catalase were consistently highest in the VYS of both species when compared to other tissues of the embryo. These activities were highly significant in the 6-12 somite VYS. ADH1 activities were significantly higher in embryos when compared to VYS in both species, except for a 27% lower activity in the early 8-10 somite mouse embryo. Mouse ADH1 activities in the VYS were significantly lower throughout the organogenesis period when compared to the rat VYS or embryos of either species. Mouse activities were lower overall in specific tissues of the embryo but maintained the same relative proportions as in the rat. ADH3 activities in the rat VYS were significantly higher by 20% than those in the mouse. Mouse embryo ADH3 activities were slow to mature, starting at a level 42% below rat, and failed to reach optimal levels until the 14-16-somite stage. Heart ADH3 activities were also significantly lower in the mouse embryo at the 7-12-somite stage. Both species have lower ADH3 activities in the early heart, relative to other embryonic tissues. These results show a more slowly maturing capacity of the mouse embryo to remove HCHO, which provides a rationale for increased sensitivity of this species to CH(3)OH-induced embryotoxicity and teratogenicity.

Alcohol Dehydrogenase↗
PubMed2000

Mitochondrial or cytosolic catalase reverses the MnSOD-dependent inhibition of proliferation by enhancing respiratory chain activity, net ATP production, and decreasing the steady state levels of H(2)O(2).

Manganese superoxide dismutase (MnSOD) overexpression has been shown to reverse the malignant phenotype in a variety of tumor cell lines. The inhibition of proliferation and reversal of the malignant phenotype has been attributed to an increase in H(2)O(2) production as a result of the dismutation reaction. However, direct evidence in support of this hypothesis has not been forthcoming. To evaluate the contribution of H(2)O(2) in the regulation of cell growth in response to MnSOD overexpression, control and MnSOD-overexpressing HT-1080 fibrosarcoma cells were transfected with constructs that direct catalase to either the mitochondrial or cytosolic compartments. Overexpression of catalase in either compartment reversed the proliferative and clonogenic inhibition associated with MnSOD overexpression, blocked the increase in the steady state levels of H(2)O(2) as measured by flow cytometric analysis of 2', 7'-dichlorofluorescein diacetate, and increased protection from the cytotoxicity of H(2)O(2). In addition, mitochondrial or cytosolic catalase enhances respiration through complex I and II in both control and MnSOD overexpressing cell lines and reverses a MnSOD-dependent decrease in net ATP production. Thus, catalase reverses the proliferative inhibition associated with MnSOD overexpression and may also play an important role in metabolic regulation.

Adenosine Triphosphate↗
PubMed2003

Ectopic expression of catalase in Drosophila mitochondria increases stress resistance but not longevity.

The goal of this study was to test the hypothesis that the rate of mitochondrial oxidant production governs the aging process of the fruit fly, Drosophila melanogaster. Catalase, an antioxidative enzyme expressed in the cytosol and peroxisomes of Drosophila, was targetted ectopically to the mitochondrial matrix by fusion of a leader peptide derived from ornithine aminotransferase with its N-terminus. The presence of the transgene encoding this fusion protein was associated with moderate (35 +/- 13%) increases in total catalase activity in most lines, and measurable levels of catalase activity in the mitochondria (30-140 U/mg protein). There was no impact on the life span of the flies at 25 degrees C, even in an exceptional line with a 149% increase in total catalase activity, and there was a small decrease in longevity at 29 degrees C. There were no compensatory changes in the rate of metabolism or physical activity, or in the levels of other major antioxidants, suggesting that the aging process was largely unaffected. Resistance to exogenous hydrogen peroxide, paraquat, and cold stress was enhanced, but there was no appreciable effect on resistance to hyperoxia. The results demonstrate the importance of mitochondrial antioxidant levels in the resistance to oxidative stress at the organismal level, and illustrate that different effects on aging and stress resistance may ensue from a single treatment. The main inferences drawn are that: (i) levels of stress resistance may neither be a cause nor a reliable indicator of the rate of aging, and (ii) bolstering antioxidant levels in Drosophila may not delay or slow down the aging process.

Animals↗
PubMed2003

Sequential inactivation of reactive oxygen species by combined overexpression of SOD isoforms and catalase in insulin-producing cells.

Insulin-producing cells show very low activity levels of the cytoprotective enzymes catalase, glutathione peroxidase, and superoxide dismutase. This weak antioxidative defense status has been considered a major feature of the poor resistance against oxidative stress. Therefore, we analyzed the protective effect of a combined overexpression of Cu,ZnSOD or MnSOD together with different levels of catalase. Catalase alone was able to increase the resistance of transfected RINm5F insulin-producing tissue culture cells against H(2)O(2) and HX/XO, but no protection was seen in the case of menadione. In combination with an increase of the MnSOD or Cu,ZnSOD expression, the protective action of catalase overexpression could be further increased and extended to the toxicity of menadione. Thus, optimal protection of insulin-producing cells against oxidative stress-mediated toxicity requires a combined overexpression of both superoxide- and hydrogen peroxide-inactivating enzymes. This treatment can compensate for the constitutively low level of antioxidant enzyme expression in insulin-producing cells and may provide an improved protection in situations of free radical-mediated destruction of pancreatic beta cells in the process of autoimmune diabetes development.

Animals↗
PubMed1998

Catalase-positive Eikenella corrodens and Eikenella-like isolates of human and canine origin.

Ten catalase-positive isolates and one catalase-negative isolate that had been assigned to Eikenella corrodens were compared to the nomenclatural type strain regarding selected phenotypic and molecular features and chromosomal deoxyribonucleic acid (DNA) relatedness using the spectrophotometric method. Five catalase-positive human isolates were assigned to the genomic species Eikenella corrodens on the basis of high DNA relatedness levels. Three others, among them strain Chen UB 204, exhibited only moderate degrees of DNA relatedness to the type strain and with each other. Two catalase-positive isolates from dogs were closely interrelated, but yielded only low degrees of DNA binding with Eikenella corrodens and the Eikenella-like human isolates. These findings confirm that the human eikenellas comprise more than one genomic species and that the canine strains represent a distinct taxonomic entity. The differentiation of the strains investigated by conventional phenotypic features, hydrolytic enzyme reactions, and cellular carbohydrate patterns was considered.

Animals↗
PubMed2003

DNA damage induced by indirect and direct acting mutagens in catalase-deficient transgenic tobacco. Cellular and acellular Comet assays.

We have measured the level of DNA damage induced by treating roots (cellular Comet assay) and isolated root nuclei (acellular Comet assay) of catalase-deficient (CAT1AS) and wild-type (SR1) tobacco with the promutagen o-phenylenediamine (o-PDA) and the direct acting genotoxic agents hydrogen peroxide and ethyl methanesulphonate (EMS). The roots of CAT1AS have about 60% less catalase activity compared to the roots of SR1. The promutagen o-PDA applied on tobacco roots induced significantly higher levels of DNA damage in the CAT1AS transgenic line than in SR1, while after application of o-PDA on isolated root nuclei, no DNA damage could be detected. In the catalase-deficient line CAT1AS about six-fold lower concentrations of H(2)O(2) are sufficient to induce the same levels of DNA damage as in SR1. By contrast, after treatment of isolated root nuclei with H(2)O(2) no difference in the induced levels of DNA damage was observed between CAT1AS and SR1. The DNA damaging effect of EMS was not affected by the presence of catalase in the tobacco roots and the levels of DNA damage measured by the cellular and acellular assay were similar. Comparing the effects of genotoxic agents in both the cellular and acellular Comet assays may help to elucidate their mechanism of action. Differences in both systems may reveal the participation of scavengers and of repair and metabolic enzymes on the activity of the genotoxic agent and the role of the cell wall in preventing the agent from reacting with nuclear DNA.

Catalase↗
PubMed2003

Infections with Haemophilus species in chronic granulomatous disease: insights into the interaction of bacterial catalase and H2O2 production.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes are incapable of generating bactericidal-reactive oxygen derivatives. Typically these patients are susceptible to life-threatening infections with catalase-producing organisms. Haemophilus species, particularly H. paraphrophilus, are not associated with CGD infections, because these organisms rarely if ever produce catalase. Haemophilus species are part of the indigenous oral microbial flora and, other than H. influenzae, are rarely recognized as pathogens. They are fastidious and require additional growth factors and capnophilic culture conditions for optimal growth and identification. Here we describe three cases of infection with non-H. influenzae (NHI) Haemophilus species in CGD patients. These organisms were catalase-negative and therefore not expected to be virulent in CGD patients, but they were also H(2)O(2) production-negative, thereby negating the putative loss of virulence of being catalase-negative. These are the first reports of NHI Haemophilus species in CGD and reinforce the critical need for careful microbiologic evaluation of infections in CGD patients.

Adult↗
PubMedDate not supplied

Prolonged oxygen-carrying ability of hemoglobin vesicles by coencapsulation of catalase in vivo.

Hemoglobin (Hb) vesicles (particle diameter, ca. 250 nm) have been developed as Hb-based oxygen carriers in which a purified Hb solution is encapsulated with a phospholipid bilayer membrane. The oxidation of Hb to nonfunctional ferric Hb (metHb) was caused by reactive oxygen species, especially hydrogen peroxide (H(2)O(2)), in vivo in addition to autoxidation. We focused on the enzymatic elimination of H(2)O(2) to suppress the metHb formation in the Hb vesicles. In this study, we coencapsulated catalase with Hb within vesicles and studied the rate of metHb formation in vivo. The Hb vesicles containing 5.6 x 10(4) unit mL(-1) catalase decreased the rate of metHb formation by half in comparison with Hb vesicles without catalase. We succeeded in prolonging the oxygen-carrying ability of the Hb vesicle in vivo by the coencapsulation of catalase.

Animals↗
PubMed2000

Modulation of the activities of catalase-peroxidase HPI of Escherichia coli by site-directed mutagenesis.

Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction which has been implicated in the activation of the antitubercular drug isoniazid in Mycobacterium tuberculosis. Hydroperoxidase I of Escherichia coli encoded by katG is a catalase-peroxidase, and residues in its putative active site have been the target of a site directed-mutagenesis study. Variants of residues R102 and H106, on the distal side of the heme, and H267, the proximal side ligand, were constructed, all of which substantially reduced the catalatic activity and, to a lesser extent, the peroxidatic activity. In addition, the heme content of the variants was reduced relative to the wild-type enzyme. The relative ease of heme loss from HPI and a mixture of tetrameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry analysis. Conversion of W105 to either an aromatic (F) or aliphatic (I) residue caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity. The peroxidatic-to-catalatic ratio of the W105F variant was increased 2800-fold such that compound I could be identified by both electronic and EPR spectroscopy as being similar to the porphyrin cation radical formed in other catalases and peroxidases. Compound I, when generated by a single addition of H(2)O(2), decayed back to the native or resting state within 1 min. When H(2)O(2) was generated enzymatically in situ at low levels, active compound I was evident for up to 2 h. However, such prolonged treatment resulted in conversion of compound I to a reversibly inactivated and, eventually, to an irreversibly inactivated species, both of which were spectrally similar to compound I.

Amino Acid Substitution↗
PubMed1995

Electron tunneling and ab initio calculations related to the one-electron oxidation of NAD(P)H bound to catalase.

Models for NAD(P)H le- oxidation in bovine catalase were studied using Hartree-Fock ab initio calculations, along with information taken from the published X-ray structure of the enzyme. Geometries and energies of ground states and transition states were calculated at the 6-31G* level for N-methyl-1,4-dihydropyridine and N-methyl-1,4-dihydronicotinamide undergoing the pathway (i) le- oxidation to yield the radical cation, (ii) general-base-catalyzed (hydroxide and/or imidazole) deprotonation of the radical cation to yield the neutral radical, and (iii) le- oxidation of the neutral radical to the N-methylpyridinium or N-methylnicotinamide cation. Barrier heights for deprotonation of the radical cation intermediates were calculated to be 7-11 kcal/mol. Kinetic isotope effects were calculated for general-base-catalyzed deprotonation of the N-methyl-1,4-dihydropyridine radical cation and the 4,4-dideuterio species and found to be kH/kD2 = 5.38 (hydroxide) or 3.64 (imidazole), in qualitative agreement with published experimental isotope effects for the analogous deprotonation of N-methyl-1,10-dihydroacridan or the N-methyl-1,10-dideuterioacridan radical cation. In the calculated transition state for imidazole deprotonation of the N-methyl-1,4-dihydronicotinamide radical cation, an unusual short contact was calculated and interpreted as a hydrogen bond (2.35 A) between the amide oxygen and the hydrogen attached to C2 of imidazole. Similar hydrogen bonds were also observed and calculated at the 3-21G and 6-31G* levels between His234 of catalase and the amide oxygen of bound NAD(P)H and complexes of N,N'-dimethyl-1,4-dihydronicotinamide or cis-N-methylformamide with N-methylimidazole. Comparison of these results to the X-ray structure of bovine catalase allows for further interpretation of the possible roles of the imidazole bases His234 and His304 and the hydrogen-bonded contacts in the NAD(P)H binding site. Electron tunneling pathways between NAD(P)H and the iron protoporphyrin IX (PP-IX) axial tyrosinate ligand Tyr357 in molecular dynamics and X-ray crystal structures of bovine catalase were calculated using PATHWAYS II (version 2.01). The pathways which were calculated included those involving the amino acid residue Tyr214, which is near the NAD(P)H binding site. Coupling involving Tyr357 was not particularly efficient; however, strong coupling between Tyr214 and iron-protoporphyrin IX was observed. These pathways may be important if electron transfer is stepwise; i.e., Tyr214 oxidized first, followed by NAD(P)H.

Amino Acid Sequence↗
PubMed1993

The dimanganese(III,IV) oxidation state of catalase from Thermus thermophilus: electron nuclear double resonance analysis of water and protein ligands in the active site.

The 1H hyperfine tensors of the dimanganese(III,IV) oxidation state of the non-heme-type catalase enzyme from the thermophilic bacterium Thermus thermophilus have been measured by electron nuclear double resonance (ENDOR) spectroscopy at pH 6.5-9. These were compared to model dimanganese(III,IV) complexes possessing six-coordinate N4O2, N3O3, and O6 atom donor sets to each Mn and mu-oxo and mu-carboxylato bridging ligands. The lack of 14N hyperfine couplings in the enzyme suggests either O6 or O5N ligand donors to each Mn. Moreover, the two sigma coordination sites on Mn(III) directed at the dz2 orbital cannot be occupied by N ligands. The 1H ENDOR spectrum revealed two types of anisotropic tensors, attributable to two D2O-exchangeable protons on the basis of the magnitude of the electron paramagnetic resonance (EPR) line narrowing in D2O. All six of the 1H hyperfine couplings are proposed to arise from a single displaceable water molecule in the active site, on the basis of their reversible disappearance, upon incubation in D2O or by precipitation from ammonium sulfate, and by simulation of the 1H ENDOR spectrum. The Mn ions are coordinated predominantly by nonmagnetic O atoms lacking covalently bound protons in both alpha and beta positions. This implicates predominantly carboxylato-type ligands (Asp and Glu) and possibly a di-mu-oxo bridge between Mn ions. The latter is supported also by the presence of strong antiferromagnetic coupling. Comparison to other dimetalloproteins also possessing the four-helix bundle structural motif shows that the polyoxo(carboxylato) coordination in catalase differs significantly from the polyhistidine coordination adopted by the diiron(II,II) site in the O2-binding protein myohemerythrin, but resembles the polycarboxylato ligation adopted by the diiron(III,III) site of ribonucleotide reductase. The catalase 1H ENDOR spectrum is essentially identical to that for the exchangeable protons in the active site of the diiron(II,III) state of uteroferrin, an acid phosphatase [Doi et al. (1988) J. Biol. Chem. 263, 5757-5763], and also for a polycarboxylato complex possessing the Mn2(mu-O)2 core with H-bonded water ligands. The 1H ENDOR line shape in catalase could be simulated using a theoretical model suitable for multispin clusters. It treats the two Mn spins as point dipoles which are exchange-coupled. It includes both dipolar and isotropic ligand hyperfine couplings. Using this model, the position of the proton with the largest interaction could be located with respect to the Mn-Mn vector because of the extreme sensitivity of line shape to position.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗
PubMed2004

Kinetic evaluation of catalase and peroxygenase activities of tyrosinase.

Tyrosinase is a copper monooxygenase containing a coupled dinuclear copper active site (type-3 copper), which catalyzes oxygenation of phenols (phenolase activity) as well as dehydrogenation of catechols (catecholase activity) using O(2) as the oxidant. In this study, catalase activity (conversion of H(2)O(2) to (1/2)O(2) and H(2)O) and peroxygenase activity (H(2)O(2)-dependent oxygenation of substrates) of mushroom tyrosinase have been examined kinetically by using amperometric O(2) and H(2)O(2) sensors. The catalase activity has been examined by monitoring the initial rate of O(2) production from H(2)O(2) in the presence of a catalytic amount of tyrosinase in 0.1 M phosphate buffer (pH 7.0) at 25 degrees C under initially anaerobic conditions. It has been found that the catalase activity of mushroom tyrosinase is three-order of magnitude greater than that of mollusk hemocyanin. The higher catalase activity of tyrosinase could be attributed to easier accessibility of H(2)O(2) to the dinuclear copper site of tyrosinase. Mushroom tyrosinase has also been demonstrated for the first time to catalyze oxygenation reaction of phenols with H(2)O(2) (peroxygenase activity). The reaction has been investigated kinetically by monitoring the H(2)O(2) consumption rate in 0.5 M borate buffer (pH 7.0) under aerobic conditions. Similarity of the substituent effects of a series of p-substituted phenols in the peroxygenase reaction with H(2)O(2) to those in the phenolase reaction with O(2) as well as the absence of kinetic deuterium isotope effect with a perdeuterated substrate (p-Cl-C(6)D(4)OH vs p-Cl-C(6)H(4)OH) clearly demonstrated that the oxygenation mechanisms of phenols in both systems are the same, that is, the electrophilic aromatic substitution reaction by a (micro-eta(2):eta(2)-peroxo)dicopper(II) intermediate of oxy-tyrosinase.

Agaricus↗
PubMed2005

Role of the Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG) as revealed by KatG(M255I).

Catalase-peroxidases (KatGs) are bifunctional enzymes possessing both catalase and peroxidase activities. Four crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link which has been suggested to impart catalatic activity to the KatGs. To decipher the individual roles of the two cross-links in the Met-Tyr-Trp adduct, we have focused on recombinant Mycobacterium tuberculosis KatG(M255I). UV-visible spectroscopic and mass spectrometric studies of the peptide fragments resulting from tryptic digestion of KatG(M255I) confirmed the presence of the single Tyr-Trp cross-link, as well as a 2e- oxidized form which is postulated to be an intermediate generated during Met-Tyr-Trp cross-link formation. KatG(M255I) lacking the Tyr-Trp cross-link was also prepared, and incubation with peroxyacetic acid, but not 2-methyl-1-phenyl-2-propyl hydroperoxide, resulted in complete formation of the Tyr-Trp cross-link. A mechanism for Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies with KatG(M255I) were performed, allowing characterization of compounds I, II, and III. Interestingly, two compound II intermediates were identified: (KatG*)(Por)Fe(III)-OH, where KatG* represents a protein-based radical, and oxoferryl (KatG)(Por)Fe(IV)=O. Insight into the contributions of the individual Tyr-Trp and Met-Tyr cross-links to catalase activity is presented, as is the overall contribution of the Met-Tyr-Trp cross-link to the structure-function-spectroscopy relationship and catalase-peroxidase mechanism in KatG.

Amino Acid Sequence↗
PubMed1999

Spectral and kinetic studies of the oxidation of monosubstituted phenols and anilines by recombinant Synechocystis catalase-peroxidase compound I.

A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Synechocystis PCC 6803 is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the first examination of second-order rate constants for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by a bifunctional catalase-peroxidase compound I using the sequential-mixing stopped-flow technique. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. A >/=50-fold excess of peroxoacetic acid is required to obtain a spectrum of relatively pure and stable compound I which is characterized by about 40% hypochromicity, a Soret maximum at 406 nm, and isosbestic points between the native enzyme and compound I at 357 and 430 nm. The apparent second-order rate constant for formation of compound I from ferric enzyme and peroxoacetic acid is (8.74 +/- 0.26) x 10(3) M(-)(1) s(-)(1) at pH 7. 0. Reduction of compound I by aromatic donor molecules is dependent upon the substituent effect on the benzene ring. The apparent second-order rate constants varied from (3.6 +/- 0.1) x 10(6) M(-)(1) s(-)(1) for p-hydroxyaniline to (5.0 +/- 0.1) x 10(2) M(-)(1) s(-)(1) for p-hydroxybenzenesulfonic acid. They are shown to correlate with the substituent constants in the Hammett equation, which suggests that in bifunctional catalase-peroxidases the aromatic donor molecule donates an electron to compound I and loses a proton simultaneously. The value of rho, the susceptibility factor in the Hammett equation, is -3.4 +/- 0.4 for the phenols and -5.1 +/- 0.8 for the anilines. The pH dependence of compound I reduction by aniline exhibits a relatively sharp maximum at pH 5. The redox intermediate formed upon reduction of compound I has spectral features which indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.

Aniline Compounds↗
PubMedDate not supplied

Purification of a catalase from Thermus thermophilus via IMAC chromatography: effect of the support.

A hexameric Mn-catalase was purified from crude extracts of Thermus thermophilus using ammonium sulfate precipitation and ion metal-chelate affinity chromatography (IMAC). Eupergit 250 and Sepabeads FP-EP3 epoxy supports derivatized with iminodiacetic acid (IDA) and copper were used, at similar micromole/packed milliliter of support. Although Eupergit 250-IDA-Cu support adsorbed 80% of the total proteins in the extract, it exhibited a minimum affinity for the catalase. On the other hand, Sepabeads FP-EP3-IDA-Cu allowed the full adsorption of the catalase activity, which could be desorbed in fractions of different purity. This was attributed to a different geometrical congruence of the support surfaces with the enzyme surface, resulting in a different ability to form multipoint interactions with the proteins. Thus, by a cleanup step, followed by a negative chromatographic step using Eupergit 250-IDA-Cu2+ and by the adsorption of the catalase on Sepabeads-IDA-Cu2+ support, a pure enzyme fraction was obtained and its N-terminal end was sequenced.

Adsorption↗
PubMed2005

Mushroom tyrosinase: catalase activity, inhibition, and suicide inactivation.

Mushroom tyrosinase exhibits catalase activity with hydrogen peroxide (H(2)O(2)) as substrate. In the absence of a one-electron donor substrate, H(2)O(2) is able to act as both oxidizing and reducing substrate. The kinetic parameters V(max) and K(m) that characterize the reaction were determined from the initial rates of oxygen gas production (V(0)(O)()2) under anaerobic conditions. The reaction can start from either of the two enzyme species present under anaerobic conditions: met-tyrosinase (E(m)) and deoxy-tyrosinase (E(d)). Thus, a molecule of H(2)O(2) can reduce E(m) to E(d) via the formation of oxy-tyrosinase (E(ox)) (E(m) + H(2) <==> O(2) right harpoon over left harpoon E(ox)), E(ox) releases oxygen into the medium and is transformed into E(d), which upon binding another molecule of H(2)O(2) is oxidized to E(m). The effect of pH and the action of inhibitors have also been studied. Catalase activity is favored by increased pH, with an optimum at pH = 6.4. Inhibitors that are analogues of o-diphenol, binding to the active site coppers diaxially, do not inhibit catalase activity but do reduce diphenolase activity. However, chloride, which binds in the equatorial orientation to the protonated enzyme (E(m)H), inhibits both catalase and diphenolase activities. Suicide inactivation of the enzyme by H(2)O(2) has been demonstrated. A kinetic mechanism that is supported by the experimental results is presented and discussed.

Agaricales↗