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PubMed2003

The Pseudomonas secretory product pyocyanin inhibits catalase activity in human lung epithelial cells.

Pyocyanin, produced by Pseudomonas aeruginosa, has many deleterious effects on human cells that relate to its ability to generate reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Human cells possess several mechanisms to protect themselves from ROS, including manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), and catalase. Given the link between pyocyanin-mediated epithelial cell injury and oxidative stress, we assessed pyocyanin's effect on MnSOD, CuZnSOD, and catalase levels in the A549 human alveolar epithelial cell line and in normal human bronchial epithelial cells. In both cell types, CuZnSOD and MnSOD were unaltered, but over 24 h pyocyanin significantly decreased cellular catalase activity and protein content. Pyocyanin also decreased catalase mRNA. Overexpression of MnSOD in A549 cells prevented pyocyanin-mediated loss of catalase protein, but catalase activity still declined. Furthermore, pyocyanin decreased catalase activity, but not protein, in A549 cells overexpressing human catalase. These data suggest a direct effect of pyocyanin on catalase activity. Addition of pyocyanin to catalase in a cell-free system also decreased catalase activity. Mammalian catalase binds four NADPH molecules, helping maintain enzyme activity. Spin-trapping data suggest that pyocyanin directly oxidizes this NADPH, producing superoxide. We conclude that pyocyanin may decrease cellular catalase activity via both transcriptional regulation and direct inactivation of the enzyme. Decreased cellular catalase activity and failure to augment MnSOD could contribute to pyocyanin-dependent cytotoxicity.

Binding Sites↗
PubMed2005

Simple PCR heteroduplex, SSCP mutation screening methods for the detection of novel catalase mutations in Hungarian patients with type 2 diabetes mellitus.

BACKGROUND: The enzyme catalase is the main regulator of hydrogen peroxide metabolism. Deficiency of catalase may cause high concentrations of hydrogen peroxide and increase the risk of the development of pathologies for which oxidative stress is a contributing factor, for example, type 2 diabetes mellitus. Catalase deficiency has been reported to be associated with increased frequency of diabetes mellitus in a cohort of patients in Hungary. In this cohort, the majority of mutations in the catalase gene occur in exon 2. METHODS: Type 2 diabetic patients (n=308) were evaluated for mutations in intron 1 (81 bp), exon 2 (172 bp) and intron 2 (13 bp) of the catalase gene. Screening for mutations utilized PCR single-strand conformational polymorphism (SSCP) and PCR heteroduplex methods. Verification of detected mutations was by nucleotide sequence analysis. RESULTS: A total of 11 catalase gene mutations were detected in the 308 subjects (3.57%, p<0.001). Five of the 11 were at two previously reported mutation sites: exon 2 (79) G insertion and (138) GA insertion. Six of the 11 were at five previously unreported catalase mutation sites: intron 1 (60) G-->T; intron 2 (7) G-->A and (5) G-->C; exon 2 (96) T-->A; and exon 2 (135) T-->A. The novel missense mutations on exon 2 (96 and 135) are associated with 59% and 48% decreased catalase activity, respectively; the novel G-->C mutation on intron 2 (5) is associated with a 62% decrease in catalase activity. Mutations detected on intron 1 (60) and intron 2 (7) showed no change in catalase activity. The G-->C mutation on intron 2 (5) might be a splicing mutation. The two missense mutations on exon 2 (96) and (135) cause substitutions of amino acids 53 (Asp-->Glu) and 66 (Glu-->Cys) of the catalase protein. These are close to amino acids that are important for the binding of heme to catalase, 44 (Val) and 72-75 (Arg, Val, Val, His). Changes in heme binding may be responsible for the activity losses. CONCLUSION: Mutations that cause decreased catalase activity may contribute to susceptibility to inherited type 2 diabetes mellitus. Exon 2 and neighboring introns of the catalase gene may be minor hot spots for type 2 diabetes mellitus susceptibility mutations.

Acatalasia↗
PubMed1999

Oxidation of human catalase by singlet oxygen in myeloid leukemia cells.

Catalases are oxidized by singlet oxygen giving rise to more acidic conformers detected in zymograms after electrophoresis in polyacrylamide gels. This shift in catalase mobility can be indicative of singlet oxygen production in vivo. Catalase from human cells, as from many organisms, is susceptible to in vitro modification by singlet oxygen. Human myeloid leukemia (U937) cells were treated under different stress conditions and catalase activity and its electrophoretic mobility was monitored. The U937 cells were found to have high levels of catalase activity, as compared to cultured fibroblasts, and to be very resistant to oxidative stress. Hydrogen peroxide did not modify the electrophoretic mobility of catalase, even at doses that produced cell damage. Conditions that primarily generate superoxide, such as treatment with paraquat or heat shock, also failed to modify the enzyme. In contrast, photosensitization reactions using rose Bengal gave rise to a more acidic conformer of catalase. Singlet oxygen quenchers prevented catalase modification by rose Bengal and light. The growth medium had a photosensitizing activity. Catalase was not modified in cells illuminated in phosphate buffer but was modified in cells illuminated in phosphate buffer containing riboflavin. Intense light per se also generated a slight shift in the electrophoretic mobility of catalase. Ultraviolet light (350 or 366 nm) did cause a change in catalase, but to a less acidic catalase conformer, indicating other modifications of the enzyme. The main effect of photosensitization with methylene blue was crosslinking of the enzyme, although some shift to acidic conformers was observed at a low concentration of the photoactive compound. Results indicate that catalase can be modified by singlet oxygen generated intracellularly, even though the enzyme is predominantly inside peroxisomes. Under some photosensitization conditions, catalase modification can be used as a marker to detect intracellular singlet oxygen.

Animals↗
PubMed1979

Electron spin resonance study of the role of NO . catalase in the activation of guanylate cyclase by NaN3 and NH2OH. Modulation of enzyme responses by heme proteins and their nitrosyl derivatives.

The role of NO . catalase in the activation of partially purified soluble guanylate cyclase of rat liver by NaN3 and NH2OH was examined by electron spin resonance (ESR) spectroscopy. Equilibration of bovine liver catalase with NO resulted in formation of a paramagnetic species exhibiting a three-line ESR spectrum similar to that of NO . catalase. This paramagnetic complex produced concentration-dependent stimulation of preparations of partially purified guanylate cyclase that were devoid of detectable endogenous heme content. The stimulation of partially purified guanylate cyclase by NO . catalase was similar to that obtained with NO . hemoglobin and with NO . cytochrome P-420 prepared by reaction of hepatic microsomes of phenobarbital-treated rats with NO. By contrast, these same enzyme preparations did not respond to NO or catalase alone. Addition of hematin or hemoglobin plus a reducing agent to purified guanylate cyclase restored enzyme responsiveness to NO and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to NaN3 or NH2OH. Responses to the latter agents were restored by catalase and potentiated by a H2O2-generating system. Formation of the NO . catalase complex was evident by ESR spectroscopy in test solutions containing NaN3 or nh2oh, catalase, and a glucose-glucose oxidase, H2O2-generating system. The presence of NO . catalase correlated well with the ability of test solutions to activate purified guanylate cyclase. These results provide evidence for catalase-dependent NO generation from NaN3 and NH2OH under conditions leading to guanylate cyclase activation. Preformed NO . hemoglobin or NO . cytochrome P-420 also activated heme-deficient partially purified guanylate cyclase. The ability of several preformed NO . heme protein complexes, but not NO, to stimulate heme-deficient guanylate cyclase supports the concept that formation of the paramagnetic nitrosyl . heme complex, mediated by either enzymatic or nonenzymatic reactions, is a common and essential step in the process by which NO or NO-forming compounds activate guanylate cyclase. In the absence of the NO ligand, both hemoglobin and catalase suppress the stimulatory effects of the corresponding NO . heme proteins on guanylate cyclase. Release of each heme protein from the NO . heme protein complex occurs more rapidly under aerobic compared to anaerobic conditions. However, hemoglobin is approximately 2000 times more effective as an inhibitor of NO . hemoglobin stimulation of guanylate cyclase than is catalase as an inhibitor of NO . catalase action. This finding may explain the more pronounced decline in the rate of cGMP generation in air in the presence of NO . hemoglobin compared to NO . catalase. The results imply that guanylate cyclase responses to activators that can form NO are determined by both the stimulatory activity of the endogenous heme acceptors of NO and the relative inhibitory effects of the unliganded heme proteins present.

Anaerobiosis↗
PubMed1999

Decrease of hepatic catalase level by treatment with diallyl sulfide and garlic homogenates in rats and mice.

Diallyl sulfide (DAS) is a flavor compound derived from garlic and is active in the inhibition of chemically induced cytotoxicity and carcinogenicity in animal models. This study was conducted to examine the effects of the treatment of DAS and garlic homogenates on the activities of catalase, glutathione peroxidase, and superoxide dismutase. Male Sprague-Dawley rats were treated with DAS i.g. at daily doses of 50 or 200 mg/kg for 8 days, causing the hepatic catalase activity to decrease by 55 and 95%, respectively. Such a decrease in hepatic catalase activity was also observed when the DAS treatment was extended to 29 days. Western blot analysis showed that the DAS treatments resulted in corresponding decreases in the liver catalase protein level. No significant change in the catalase activity in the kidney, lung, and brain was observed with the treatments, but a slight decrease in heart catalase activity was observed. These treatments did not cause significant changes in superoxide dismutase and glutathione peroxidase activities in these tissues. Treatment with DAS at a daily dose of 200 mg/kg for 1-7 days resulted in a gradual decrease in the liver catalase activity to 5% of the control level, but it did not decrease the erythrocyte catalase activity. Treatment of rats with fresh garlic homogenates (2 or 4 g/kg, i.g., daily for 7 days) caused a 35% decrease in liver catalase activity. A/J mice treated with DAS and garlic homogenates also showed a decrease in the liver catalase activity. Diallyl sulfone (DASO2), a DAS metabolite, however, did not effectively decrease catalase activity in mice. The catalase activity was not inhibited by either DAS or DASO2 in vitro. The present results demonstrate that treatment with DAS and garlic homogenates decrease the hepatic catalase level in rats and mice.

Allyl Compounds↗
PubMed1996

Identification of a novel growth-promoting factor with a wide target cell spectrum from various tumor cells as catalase.

We have previously reported the purification from human erythrocyte extracts of a novel growth-promoting factor with a wide target cell spectrum. The factor has been identified as catalase. As cell extracts from a variety of tumor cell types exhibited both growth-promoting and catalase activities, the relationship between the two activities was examined using cell extracts from three different cell types, human myeloid cells (U937), human melanoma cells (A375-C6), and human B cells (Daudi). The growth-promoting and catalase activities were well correlated in these cell extracts. The antibody against human catalase absorbed not only catalase activity, but also the growth-promoting activity of extracts from these cell types. Treatment of the cell extracts from these cells with an irreversible catalase inhibitor, aminotriazole, abolished both the catalase and growth-promoting activities. In contrast, glutathione peroxidase (GSH-Px) activity was neither absorbed with the anti-catalase antibody, nor inhibited by aminotriazole. In addition, GSH-Px exhibited growth-promoting activity only in the presence of glutathione (GSH). These results, in conjunction with the effect of aminotriazole on the growth-promoting activity of catalase, suggest that catalase is the major growth-promoting molecule in the cell extracts, and H2O2-decomposing activity is important. Northern blot analysis revealed that these cells contained authentic catalase mRNA, and the mRNA level was compatible with the catalase and growth-promoting activities in the cell extracts. These results suggest that the growth-promoting activity in the tumor cell extracts is due to catalase.

Amitrole↗
PubMed2003

Catalases of Aspergillus fumigatus.

Upon infection of a host, the pathogenic fungus Aspergillus fumigatus is attacked by the reactive oxygen species produced by phagocytic cells. Detoxification of hydrogen peroxide by catalases was proposed as a way to overcome this host response. A. fumigatus produces three active catalases; one is produced by conidia, and two are produced by mycelia. The mycelial catalase Cat1p was studied previously. Here we characterized the two other catalases, their genes, and the phenotypes of gene-disrupted mutants. CatAp, a spore-specific monofunctional catalase, is resistant to heat, metal ions, and detergent. This enzyme is a dimeric protein with 84.5-kDa subunits. The 749-amino-acid polypeptide exhibits high levels of similarity to the Aspergillus nidulans CatA catalase and to bacterial catalase HPII of Escherichia coli. In spite of increased sensitivity to H(2)O(2), killing of DeltacatA conidia by alveolar macrophages and virulence in animals were similar to the killing of conidia by alveolar macrophages and virulence in animals observed for the wild type. In contrast to the Cat1p and CatAp catalases, the mycelial Cat2p enzyme is a bifunctional catalase-peroxidase and is sensitive to heat, metal ions, and detergent. This enzyme, an 82-kDa monomer, is homologous to catalase-peroxidases of several fungi and bacteria. Surprisingly, mycelium of the double Deltacat1Deltacat2 mutant with no catalase activity exhibited only slightly increased sensitivity to H(2)O(2) and was as sensitive to killing by polymorphonuclear neutrophils as mycelium of the wild-type strain. However, this mutant exhibited delayed infection in the rat model of aspergillosis compared to infection by the wild-type strain. These results indicate that conidial catalase is not a virulence factor and that mycelial catalases transiently protect the fungus from the host.

Amino Acid Sequence↗
PubMed1987

Multiple catalases in Bacillus subtilis.

Vegetative cells of Bacillus subtilis in logarithmic growth phase produced one catalase, labeled catalase 1, with a nondenatured molecular weight of 205,000. As growth progressed, other activity bands with slower electrophoretic mobilities on polyacrylamide gels appeared, including a series of bands with a common nondenatured molecular weight of 261,000, collectively labeled catalase 2, and a minor band, with a molecular weight of 387,000, labeled catalase 3. Purified spores contained only catalase 2, and it was not produced in spo0A- or spo0F-containing mutants. Strains deficient in catalase 1 or catalase 2 or both were selected after mutagenesis. Sensitivities of the two main catalases to NaCN, NaN3, hydroxylamine, and temperature were similar, but the apparent Kms for H2O2 differed, being 36.6 and 64.4 mM, respectively, for catalase 1 and catalase 2. The levels of catalase 1 increased 15-fold during growth into stationary phase and could be increased 30-fold by the addition of H2O2 to the medium. Catalase 2, which was not affected by H2O2, appeared only after the cells had reached stationary phase, and the maximum levels were only half of the basal level of catalase 1.

Bacillus subtilis↗
PubMed1992

Immunotitration of the catalase in the blood of Japanese subjects and mice suffering from acatalasemia and hypocatalasemia.

Catalase in hemolysates of normal, heterozygous hypocatalasemic and acatalasemic Japanese was immunotitrated with an anti-human blood catalase rabbit serum. Equivalence points were calculated from the regression lines between catalase activity added and catalase activity remaining in the supernatant. Catalase activities at the equivalence points of Japanese normal, hypocatalasemia and acatalasemia were similar. The results indicate that the specific activities of catalase in the normal and of the variant bloods are identical. Catalase in hemolysates of normal and variant mice was immunotitrated with an anti-mouse liver catalase rabbit serum. In contrast to Japanese acatalasemic subject, the equivalence points of catalase in heterozygous hypocatalasemic, homozygous hypocatalasemic, acatalasemic and normal hemolysates were different, and the ratios of specific activity in these variant mice to that in normal were 0.72, 0.46 and 0.21, respectively. The differences in catalase activities at equivalence points were also supported by the statistical analysis on parameters of regression lines of catalase activities remaining in the supernatant on catalase activities added in the immunotitration. These findings suggest that the molecular properties of residual catalase of Japanese acatalasemia and those of mouse acatalasemia are entirely different.

Acatalasia↗
PubMed1987

Isolation of a cDNA clone for murine catalase and analysis of an acatalasemic mutant.

We have investigated the genetic control of murine catalase expression by analyzing catalase transcription and translation products from the tissues of control (Csa) and acatalasemic (Csb) mouse strains. Csb animals possess nearly normal catalase enzyme activity levels in liver, while displaying approximately 20 and 1% of normal activity levels in kidney and red blood cells, respectively. Immunoblot analyses of catalase in these tissues have revealed reduced levels of immunologically reactive catalase protein in Csb kidney and red blood cells, paralleling the reduction of catalase enzyme activity in these tissues. In order to determine the molecular basis for Csb acatalasemia, we have isolated a cDNA clone for murine catalase and have used this probe to analyze Csa and Csb genomic DNA and catalase mRNA. These studies have revealed: 1) no restriction fragment length polymorphisms between Csa and Csb genomic DNAs; 2) no differences in the levels of Csa and Csb catalase mRNA within a single tissue; and 3) no differences in the sizes of Csa and Csb catalase mRNAs. These observations suggest that the genetic defect that produces the tissue-specific reduction of catalase expression in Csb mice is not due to a marked rearrangement of DNA within the Csb catalase structural gene. Furthermore, the Csb mutation does not act at the level of gene transcription or mRNA stability, but rather at the level of mRNA translation and/or catalase protein turnover.

Acatalasia↗
PubMed1995

Catalase-negative peroxisomes: transient appearance in rat hepatocytes during liver regeneration after partial hepatectomy.

Using light microscopy enzyme cytochemistry to localize catalase activity in peroxisomes, a population of peroxisome-negative hepatocytes was detected in livers of rats during liver regeneration induced by two-thirds partial hepatectomy. However, examination by electron microscopy revealed that this population of hepatocytes contained peroxisomes with a delimiting membrane and a nucleoid, but no cytochemically demonstrable catalase activity within their matrix. Regenerating livers 6, 18, 24, 36, 48 and 72 hours, and 1 week after partial hepatectomy showed hepatocytes without catalase activity. However, their numbers varied, with the most numerous appearing at 24 hours after partial hepatectomy. Mitosis of catalase-negative hepatocytes were seen along with mitosis of hepatocytes containing the normal complement of catalase-positive peroxisomes. The catalase-negative hepatocytes did not show evidence of apoptosis or necrotic cell death. Lysosomal acid phosphatase activity and bile canalicular ATPase activity were present in hepatocytes with catalase-negative peroxisomes. Another population of hepatocytes with a small number of catalase-positive peroxisomes appeared and were more numerous at 36 hours after partial hepatectomy; ultrastructurally, these hepatocytes contained both catalase-negative peroxisomes, which appeared to undergo dissolution, and catalase-positive peroxisomes, which were smaller in size. After complete restoration of the liver, all hepatocytes displayed essentially uniform numbers of catalase-positive peroxisomes. These studies indicated that during liver regeneration there is a transient loss of catalase in peroxisomes of some hepatocytes. These cells proliferate and with time acquire new catalase-positive peroxisomes. The observations are discussed in relation to peroxisome biogenesis, hepatocellular carcinogenesis, and oxidative stress during liver regeneration.

Animals↗
PubMed1994

Changes in the localization of catalase during differentiation of neutrophilic granulocytes.

The nature of the compartmentalization of catalase in human myeloid cells is an unresolved issue. Using a rabbit polyclonal antibody specific for catalase, indirect immunocytofluorescence of immature leukemic promyelocytes (HL-60 cells) showed a pattern of small, sharp, punctate staining in the cytoplasm of all cells, while mature neutrophils showed a larger diffuse, flocculent pattern of cytoplasmic staining. Differential centrifugation of nitrogen cavitates of HL-60 cells indicated that the putative catalase-containing compartment was relatively fragile compared with the compartment(s) that contained myeloperoxidase (MPO), beta-hexosaminidase, beta-glucuronidase, and lysosomal alpha-mannosidase activities. Parallel studies using dimethylsulfoxide (DMSO)-induced HL-60 cells and mature neutrophils showed that, in the course of differentiation, there was an apparent shift in the localization of catalase from the granule fraction to the cytosolic fraction. Percoll-sucrose density gradient centrifugation of HL-60 cell cavitates showed a catalase-containing compartment with a mean peak density (1.05 g/mL) significantly lower than that of the major myeloperoxidase-containing compartment (1.08 g/mL); in mature neutrophils, catalase activity comigrated with lactate dehydrogenase (LDH) activity. Catalase in isolated fractions was protected from proteolysis in the absence, but not in the presence, of 0.1% Triton X-100. Digitonin titration experiments confirmed the compartmentalized nature of catalase in immature HL-60 cells and were consistent with a cytosolic localization in mature neutrophils. Ultrastructural localization of catalase by Protein A-gold immunocytochemistry demonstrated four to six catalase-containing compartments in all HL-60 cell profiles. In mature neutrophils, catalase was localized primarily in the cytoplasmic matrix, although in fewer than 2% of the cell profiles, one to two catalase-containing compartments were observed. The changes in catalase localization that occur during myeloid differentiation appear to be similar to the changes that occur during erythroid and megakaryocytic differentiation, and may have potential clinical significance in the classification of acute leukemia and in the development of drug resistance.

Catalase↗
PubMedDate not supplied

Catalase mRNA expression in the male rat reproductive tract.

Reactive oxygen species (ROS) have been shown to impair sperm function. The actions of ROS are reduced by antioxidant enzymes, including catalase. Although catalase-like activity has been demonstrated in semen, there has been no localization or characterization of catalase mRNA expression in the male reproductive tract. Catalase mRNA levels were evaluated by northern blot analysis and in situ hybridization from the male reproductive organs of normal 60-day-old rats, testes of 10- to 90-day-old rats, and testes of rats subjected to efferent duct ligation. Radioactive DNA probes were synthesized using a Klenow polymerase-based specific primer synthetic procedure with a known published sequence for rat catalase. All tissues demonstrated a single transcript of 2.5 kilobases (kb). Low levels of catalase mRNA were detected in the normal testis, epididymis, vas deferens, and prostate. No expression was detectable with northern analysis in seminal vesicle. The levels of catalase mRNA in reproductive organs were compared with the high levels of expression detectable in rat liver. In the testis, catalase expression was primarily localized to peritubular and interstitial cells. In the epididymis and prostate, mRNA was detected in the epithelium. The observed decrease in catalase mRNA levels in the maturing rat testis is consistent with its interstitial localization. The increase in testicular catalase mRNA levels seen in parallel with progressive thinning of the germinal epithelium after efferent duct ligation is also in keeping with a peritubular or interstitial cell localization. The relatively low levels of catalase mRNA expression in the normal adult male reproductive tract undermine the role of catalase as a major antioxidant enzyme in these tissues. The low levels of catalase mRNA in the testis, and the undetectable levels in the seminiferous epithelium, however, imply that the germinal epithelium is predisposed to an oxidative state. These findings may help to explain the known susceptibility of the testis to oxidative stress.

Animals↗
PubMed1975

Electron paramagnetic resonance spectra of catalase in mammalian tissues.

The relatively small number of paramagnetic species and the high concentration of catalase in mammalian liver and blood make it possible to directly study this enzyme in frozen whole tissue. The EPR spectra of catalase are dependent on the heme environment and in human blood only catalase A, gxy = 6.48, 5.36 is observed whereas in liver a second spectrum, catalase B, gxy = 6.80, 5.07 can also be seen. Using rapid freeze techniques it has been shown that in rat liver catalase A corresponds to the in vivo steady state and that after death this is largely converted into catalase B. Data from the perfusion of rat livers with oxygenated and deoxygenated blood and dextran solutions together with results from in vitro studies of catalase are interpreted as indicating that catalase B results from the interaction of catalase with an organic acid, most probably formic acid, that the acid is a peroxidative substrate for catalase in vivo and that peroxidation of the acid is not the major role for catalase in rat liver. Catalase binding with other small molecules in intact liver has been demonstrated by perfusion with nitrite-containing dextrans and by intraperitoneal injection of 3-amino-1,2,4-triazole.

Amitrole↗
PubMed1986

Comparison of beef liver and Penicillium vitale catalases.

The structures of Penicillium vitale and beef liver catalase have been determined to atomic resolution. Both catalases are tetrameric proteins with deeply buried heme groups. The amino acid sequence of beef liver catalase is known and contains (at least) 506 amino acid residues. Although the sequence of P. vitale catalase has not yet been determined chemically, 670 residues have been built into the 2 A resolution electron density map and have been given tentative assignments. A large portion of each catalase molecule (91% of residues in beef liver catalase and 68% of residues in P. vitale catalase) shows structural homology. The root-mean-square deviation between 458 equivalenced C alpha atoms is 1.17 A. The dissimilar parts include a small fragment of the N-terminal arm and an additional "flavodoxin-like" domain at the carboxy end of the polypeptide chain of P. vitale catalase. In contrast, beef liver catalase contains one bound NADP molecule per subunit in a position equivalent to the chain region, leading to the flavodoxin-like domain, of P. vitale catalase. The position and orientation of the buried heme group in the two catalases, relative to the mutually perpendicular molecular dyad axes, are identical within experimental error. A mostly hydrophobic channel leads to the buried heme group. The surface opening to the channel differs due to the different disposition of the amino-terminal arm and the presence of the additional flavodoxin-like domain in P. vitale catalase. Possible functional implications of these comparisons are discussed.

Amino Acid Sequence↗
PubMed2004

Unusual Cys-Tyr covalent bond in a large catalase.

Catalase-1, one of four catalase activities of Neurospora crassa, is associated with non-growing cells and accumulates in asexual spores. It is a large, tetrameric, highly efficient, and durable enzyme that is active even at molar concentrations of hydrogen peroxide. Catalase-1 is oxidized at the heme by singlet oxygen without significant effects on enzyme activity. Here we present the crystal structure of catalase-1 at 1.75A resolution. Compared to structures of other catalases of the large class, the main differences were found at the carboxy-terminal domain. The heme group is rotated 180 degrees around the alpha-gamma-meso carbon axis with respect to clade 3 small catalases. There is no co-ordination bond of the ferric ion at the heme distal side in catalase-1. The catalase-1 structure exhibited partial oxidation of heme b to heme d. Singlet oxygen, produced catalytically or by photosensitization, may hydroxylate C5 and C6 of pyrrole ring III with a subsequent formation of a gamma-spirolactone in C6. The modification site in catalases depends on the way dioxygen exits the protein: mainly through the central channel or the main channel in large and small catalases, respectively. The catalase-1 structure revealed an unusual covalent bond between a cysteine sulphur atom and the essential tyrosine residue of the proximal side of the active site. A peptide with the predicted theoretical mass of the two bound tryptic peptides was detected by mass spectrometry. A mechanism for the Cys-Tyr covalent bond formation is proposed. The tyrosine bound to the cysteine residue would be less prone to donate electrons to compound I to form compound II, explaining catalase-1 resistance to substrate inhibition and inactivation. An apparent constriction of the main channel at Ser198 lead us to propose a gate that opens the narrow part of the channel when there is sufficient hydrogen peroxide in the small cavity before the gate. This mechanism would explain the increase in catalytic velocity as the hydrogen peroxide concentration rises.

Amino Acid Sequence↗
PubMed1973

The characteristics of the "peroxidatic" reaction of catalase in ethanol oxidation.

Ethanol oxidation by rat liver catalase (the ;peroxidatic' reaction) was studied quantitatively with respect to the rate of H(2)O(2) generation, catalase haem concentration, ethanol concentration and the steady-state concentration of the catalase-H(2)O(2) intermediate (Compound I). At a low ratio of H(2)O(2)-generation rate to catalase haem concentration, the rate of ethanol oxidation was independent of the catalase haem concentration. The magnitude of the inhibition of ethanol oxidation by cyanide was not paralleled by the formation of the catalase-cyanide complex and was altered greatly by varying either the ethanol concentration or the ratio of the rate of H(2)O(2) generation to catalase haem concentration. The ethanol concentration producing a half-maximal activity was also dependent on the ratio of the H(2)O(2)-generation rate to catalase haem concentration. These phenomena are explained by changes in the proportion of the ;catalatic' and ;peroxidatic' reactions in the overall H(2)O(2)-decomposition reaction. There was a correlation between the proportion of the ;peroxidatic' reaction in the overall catalase reaction and the steady-state concentration of the catalase-H(2)O(2) intermediate. Regardless of the concentration of ethanol and the rate of H(2)O(2) generation, a half-saturation of the steady state of the catalase-H(2)O(2) intermediate indicated that about 45% of the H(2)O(2) was being utilized by the ethanol-oxidation reaction. The results reported show that the experimental results in the study on the ;microsomal ethanol-oxidation system' may be reinterpreted and the catalase ;peroxidatic' reaction provides a quantitative explanation for the activity hitherto attributed to the ;microsomal ethanol-oxidation system'.

Animals↗
PubMed2000

Immunological detection of alkaline-diaminobenzidine-negativeperoxisomes of the nematode Caenorhabditis elegans purification and unique pH optima of peroxisomal catalase.

We purified catalase-2 of the nematode Caenorhabditis elegans and identified peroxisomes in this organism. The peroxisomes of C. elegans were not detectable by cytochemical staining using 3, 3'-diaminobenzidine, a commonly used method depending on the peroxidase activity of peroxisomal catalase at pH 9 in which genuine peroxidases are inactive. The cDNA sequences of C. elegans predict two catalases very similar to each other throughout the molecule, except for the short C-terminal sequence; catalase-2 (500 residues long) carries a peroxisomal targeting signal 1-like sequence (Ser-His-Ile), whereas catalase-1 does not. The catalase purified to near homogeneity from the homogenate of C. elegans cells consisted of a subunit of 57 kDa and was specifically recognized by anti-(catalase-2) serum but not by anti-(catalase-1) serum. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected catalase-2 inside vesicles judged to be peroxisomes using morphological criteria. The purified enzyme (220 kDa) was tetrameric, similar to many catalases from various sources, but exhibited unique pH optima for catalase (pH 6) and peroxidase (pH 4) activities; the latter value is unusually low and explains why the peroxidase activity was undetectable using the standard alkaline diaminobenzidine-staining method. These results indicate that catalase-2 is peroxisomal and verify that it can be used as a marker enzyme for C. elegans peroxisomes.

3,3'-Diaminobenzidine↗